Modeling SARS-CoV-2 propagation using rat coronavirus-associated shedding and transmission.

At present, global immunity to SARS-CoV-2 resides within a heterogeneous combination of susceptible, naturally infected and vaccinated individuals. The extent to which viral shedding and transmission occurs on re-exposure to SARS-CoV-2 is an important determinant of the rate at which COVID-19 achieves endemic stability. We used Sialodacryoadenitis Virus (SDAV) in rats to model the extent to which immune protection afforded by prior natural infection via high risk (inoculation; direct contact) or low risk (fomite) exposure, or by vaccination, influenced viral shedding and transmission on re-exposure. On initial infection, we confirmed that amount, duration and consistency of viral shedding, and seroconversion rates were correlated with exposure risk. Animals were reinfected after 3.7-5.5 months using the same exposure paradigm. 59% of seropositive animals shed virus, although at lower amounts. Previously exposed seropositive reinfected animals were able to transmit virus to 25% of naive recipient rats after 24-hour exposure by direct contact. Rats vaccinated intranasally with a related virus (Parker's Rat Coronavirus) were able to transmit SDAV to only 4.7% of naive animals after a 7-day direct contact exposure, despite comparable viral shedding. Cycle threshold values associated with transmission in both groups ranged from 29-36 cycles. Observed shedding was not a prerequisite for transmission. Results indicate that low-level shedding in both naturally infected and vaccinated seropositive animals can propagate infection in susceptible individuals. Extrapolated to COVID-19, our results suggest that continued propagation of SARS-CoV-2 by seropositive previously infected or vaccinated individuals is possible.

SDAV, the Rat Coronavirus-How Much Do We Know about It in the Light of Potential Zoonoses.

Sialodacryoadenitis virus (SDAV) is known to be an etiological agent, causing infections in laboratory rats. Until now, its role has only been considered in studies on respiratory and salivary gland infections. The scant literature data, consisting mainly of papers from the last century, do not sufficiently address the topic of SDAV infections. The ongoing pandemic has demonstrated, once again, the role of the Coronaviridae family as extremely dangerous etiological agents of human zoonoses. The ability of coronaviruses to cross the species barrier and change to hosts commonly found in close proximity to humans highlights the need to characterize SDAV infections. The main host of the infection is the rat, as mentioned above. Rats inhabit large urban agglomerations, carrying a vast epidemic threat. Of the 2277 existing rodent species, 217 are reservoirs for 66 zoonotic diseases caused by viruses, bacteria, fungi, and protozoa. This review provides insight into the current state of knowledge of SDAV characteristics and its likely zoonotic potential.

Simultaneous serodetection of major rat infectious pathogens by a multiplex immunochromatographic assay.

Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 µl PBS and either 0.75 µl of rat serum or 1.5 µl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.

Discovery, diversity and evolution of novel coronaviruses sampled from rodents in China.

Although rodents are important reservoirs for RNA viruses, to date only one species of rodent coronavirus (CoV) has been identified. Herein, we describe a new CoV, denoted Lucheng Rn rat coronavirus (LRNV), and novel variants of two Betacoronavirus species termed Longquan Aa mouse coronavirus (LAMV) and Longquan Rl rat coronavirus (LRLV), that were identified in a survey of 1465 rodents sampled in China during 2011-2013. Phylogenetic analysis revealed that LAMV and LRLV fell into lineage A of the genus Betacoronavirus, which included CoVs discovered in humans and domestic and wild animals. In contrast, LRNV harbored by Rattus norvegicus formed a distinct lineage within the genus Alphacoronavirus in the 3CL(pro), RdRp, and Hel gene trees, but formed a more divergent lineage in the N and S gene trees, indicative of a recombinant origin. Additional recombination events were identified in LRLV. Together, these data suggest that rodents may carry additional unrecognized CoVs.

Neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contribute to pathology.

Polymorphonuclear neutrophils (PMN) infiltrate the respiratory tract early after viral infection and can contribute to both host defence and pathology. Coronaviruses are important causes of respiratory tract infections, ranging from mild to severe depending on the viral strain. This study evaluated the role of PMN during a non-fatal pulmonary coronavirus infection in the natural host. Rat coronavirus (RCoV) causes respiratory disease in adult rats, characterized by an early PMN response, viral replication and inflammatory lesions in the lungs, mild weight loss and effective resolution of infection. To determine their role during RCoV infection, PMN were depleted and the effects on disease progression, viral replication, inflammatory response and lung pathology were analysed. Compared with RCoV infection in control animals, PMN-depleted rats had worsened disease with weight loss, clinical signs, mortality and prolonged pulmonary viral replication. PMN-depleted animals had fewer macrophages and lymphocytes in the respiratory tract, corresponding to lower chemokine levels. Combined with in vitro experiments showing that PMN express cytokines and chemokines in response to RCoV-infected alveolar epithelial cells, these findings support a role for PMN in eliciting an inflammatory response to RCoV infection. Despite their critical role in the protection from severe disease, the presence of PMN was correlated with haemorrhagic lesions, epithelial barrier permeability and cellular inflammation in the lungs. This study demonstrated that while PMN are required for an effective antiviral response, they also contribute to lung pathology during RCoV infection.

Rat respiratory coronavirus infection: replication in airway and alveolar epithelial cells and the innate immune response.

The rat coronavirus sialodacryoadenitis virus (SDAV) causes respiratory infection and provides a system for investigating respiratory coronaviruses in a natural host. A viral suspension in the form of a microspray aerosol was delivered by intratracheal instillation into the distal lung of 6-8-week-old Fischer 344 rats. SDAV inoculation produced a 7 % body weight loss over a 5 day period that was followed by recovery over the next 7 days. SDAV caused focal lesions in the lung, which were most severe on day 4 post-inoculation (p.i.). Immunofluorescent staining showed that four cell types supported SDAV virus replication in the lower respiratory tract, namely Clara cells, ciliated cells in the bronchial airway and alveolar type I and type II cells in the lung parenchyma. In bronchial alveolar lavage fluid (BALF) a neutrophil influx increased the population of neutrophils to 45 % compared with 6 % of the cells in control samples on day 2 after mock inoculation. Virus infection induced an increase in surfactant protein SP-D levels in BALF of infected rats on days 4 and 8 p.i. that subsided by day 12. The concentrations of chemokines MCP-1, LIX and CINC-1 in BALF increased on day 4 p.i., but returned to control levels by day 8. Intratracheal instillation of rats with SDAV coronavirus caused an acute, self-limited infection that is a useful model for studying the early events of the innate immune response to respiratory coronavirus infections in lungs of the natural virus host.

Rat coronaviruses infect rat alveolar type I epithelial cells and induce expression of CXC chemokines.

We analyzed the ability of two rat coronavirus (RCoV) strains, sialodacryoadenitis virus (SDAV) and Parker's RCoV (RCoV-P), to infect rat alveolar type I cells and induce chemokine expression. Primary rat alveolar type II cells were transdifferentiated into the type I cell phenotype. Type I cells were productively infected with SDAV and RCoV-P, and both live virus and UV-inactivated virus induced mRNA and protein expression of three CXC chemokines: CINC-2, CINC-3, and LIX, which are neutrophil chemoattractants. Dual immunolabeling of type I cells for viral antigen and CXC chemokines showed that chemokines were expressed primarily by uninfected cells. Virus-induced chemokine expression was reduced by the IL-1 receptor antagonist, suggesting that IL-1 produced by infected cells induces uninfected cells to express chemokines. Primary cultures of alveolar epithelial cells are an important model for the early events in viral infection that lead to pulmonary inflammation.

[Ultrastructural observation of rat thymus tissue with coronavirus infection].

OBJECTIVE: To investigate the ultrastructure of rat thymus tissues with rat coronavirus (RCV) infection for clarifying the mechanism responsible for the morphological changes of the cells infected by RCV. METHODS: Routine electron microscopy was performed for observing RCV-infected rat thymus tissues. RESULTS: Following RCV infection, endoplasmic reticulum (ER) pools of different dimensions were observed in the cytoplasm of the thymic epithelial reticular cells, merging subsequently with each other into larger ER lakes filled with particles of mature RCV, or viral inclusion bodies. After germination on the ER membrane, the viruses entered the matrix of the ER lake to mature and were eventually excreted to the extracellular space. The RCV particles were spherical in shape with a diameter of 100-130 nm and two distinct membranes, the outer one being the envelope and the inner one the nuclear capsid to enclose the viroplasm. Between the envelop and nuclear capsid was a electron-lucent middle layer comprising one to two thin membranous structures. Large quantity of short spike-like projections starting from the nucleus capsid penetrated the middle layer and the envelop to reach the glycoprotein coat and formed a corona-like structure. Mature RCV particles were distributed around the ER pools, cytoplasm, and intercellular space, and the RCVs in the endosome/lysosome were devoid of the envelop and nuclear capsid. CONCLUSION: The ER lakes are involved in the maturation of the viruses, and the envelop and nuclear capsid of the virus entering the cells from extracellular space are removed and degraded in the endosome/lysosome. Replications of virus occurs in plasma of the thymic epithelial reticular cells, and no RCV can be detected in the thymocytes.

Detection of rodent coronaviruses by use of fluorogenic reverse transcriptase-polymerase chain reaction analysis.

Reverse transcriptase-polymerase chain reaction (RT-PCR) assays have proved useful for the detection of mouse hepatitis virus (MHV) and rat coronavirus (RCV) in acutely infected animals and contaminated biomaterials. Fluorogenic nuclease RT-PCR assays combine RT-PCR with an internal fluorogenic hybridization probe, thereby eliminating post-PCR processing and potentially enhancing specificity. Consequently, a fluorogenic nuclease RT-PCR assay specific for rodent coronaviruses was developed. Primer and probe sequences were selected from the viral genome segment that encodes the membrane (M) protein that is highly conserved among rodent coronaviruses. Use of the fluorogenic nuclease RT-PCR detected all strains of MHV and RCV that were evaluated, but did not detect other RNA viruses that naturally infect rodents. Use of the assay detected as little as two femtograms of in vitro transcribed RNA generated from cloned amplicon, and when compared directly with mouse antibody production tests, had similar sensitivity at detecting MHV-A59 in infected cell culture lysates. Finally, use of the assay detected coronavirus RNA in tissues, cage swipes, and feces obtained from mice experimentally infected with MHV, and in tissues and cage swipes obtained from rats naturally infected with RCV. These results indicate that the fluorogenic nuclease RT-PCR assay should provide a potentially high-throughput, PCR-based method to detect rodent coronaviruses in infected rodents and contaminated biological materials.

Reverse transcriptase polymerase chain reaction-based diagnosis and molecular characterization of a new rat coronavirus strain.

BACKGROUND AND PURPOSE: Rat coronaviruses (RCV) are highly infectious and spread rapidly through laboratory rat colonies, causing sneezing, nasal and ocular discharges, photophobia, and cervical swelling. Current diagnostic methods include serologic testing and histologic examination. During a recent rat coronavirus outbreak, we tested a rapid, noninvasive method of RCV diagnosis that involved use of reverse transcriptase-polymerase chain reaction (RT-PCR) analysis to detect RCV RNA on cages housing infected rats. METHODS: The RT-PCR was used to detect RCV RNA in tissues from infected rats and on cages housing infected rats and to amplify portions of the RCV N, M, and S genes for molecular characterization. RESULTS: The RT-PCR detected RCV RNA on cages and in tissues from infected rats. The RCV-NJ N gene is most closely related to the MHV-Y N gene. The M proteins of RCV-NJ and RCV-SDA are 99% homologous, and the six RCV S protein fragments are 97 to 100% homologous. CONCLUSIONS: Use of RT-PCR with cage-swab specimens was capable of diagnosing RCV infection in and viral excretion from rats. Additionally, molecular characterization of the N, M, and S genes of RCV-NJ provided baseline information that can be used in performing further epidemiologic studies.

Comparison of the pathogenicity in rats of rat coronaviruses of different neutralization groups.

BACKGROUND AND PURPOSE: Rat coronaviruses (RCVs) are common natural pathogens of rats that cause clinical illness, necrosis, and inflammation of respiratory, salivary, and lacrimal organs. The aim of the study was to determine whether antigenically different strains of RCV vary in their pathogenic potential in rats. METHODS: Neutralization groups were identified by use of RCV strain-specific antisera. Sprague Dawley rats were inoculated oronasally with RCV-SDA, RCV-BCMM, or RCV-W. Histologic examination, immunohistochemical analysis, and reverse transcriptase-polymerase chain reaction analysis were performed on tissues from infected rats. RESULTS: Clinical illness was not evident in any of the inoculated rats. The RCV-SDA strain caused mild lesions in the exorbital gland of one rat. The RCV-BCMM strain caused severe lesions in the Harderian and parotid glands and mild lesions in the exorbital glands, lungs, and nasal mucosa. The RCV-W strain caused severe lesions in the Harderian, exorbital, and parotid glands and mild lesions in the submandibular glands, lungs, and nasal mucosa. The RNA concentration was highest in the Harderian, parotid, and exorbital glands of RCV-BCMM- and RCV-W-infected rats at postinoculation day 7. CONCLUSIONS: Although RCV-SDA, RCV-BCMM, and RCV-W caused different degrees and patterns of lesions, neutralization groups are not useful for predicting the pathogenic potential of a new RCV isolate.

Replication of rat coronaviruses in intestinal cell line, RCN-9, derived from F344 rats.

To examine the susceptibility of the epithelial cell line to rat coronavirus (RCV), we inoculated sialodacryoadenitis virus and Parker's RCV into five cell lines; JTC-19, rat L2, LLC, RCN-9 and LBC cells originating in the lungs, intestines and mammary tumors of rodents. Both RCVs were replicated in LBC and RCN-9 cells, but not in the others. The infectivity titers of both RCVs grown in RCN-9 cells were significantly higher than those in LBC cells in every passage (2.5-3.9 log rate). Both RCVs replicated in LBC cells showed higher tropism to RCN-9 cells than to LBC cells, suggesting that RCN-9 cells are more suitable for the replication of RCVs than LBC cells. The RCN-9 cell line would be useful for the investigation of RCV infection in rodents.

The murine coronavirus as a model of trafficking and assembly of viral proteins in neural tissue.

The replication of JHM, a murine coronavirus, provides a useful model of the assembly and dissemination of viral components in neuronal cells. Involvement of microtubules in virus trafficking is an important feature which may explain dissemination of the infection from primary cell targets at olfactory, hippocampal and cerebellar sites within the central nervous system, resulting in severe neuropathies.

Attachment glycoproteins and receptor specificity of rat coronaviruses.

Murine coronavirus (MHV) and rat coronavirus (RCV) are antigenically related viruses that have different natural rodent hosts. Both MHV and RCV can be propagated in the L2(Percy) and CMT-93 mouse cell lines. In these cell lines MHV uses the MHV receptor (MHVR or Bgp1a) and several related murine Bgp glycoproteins in the immunoglobulin superfamily as receptors. To determine whether RCV also uses these murine glycoproteins as receptors, we characterized the envelope glycoproteins of two strains of RCV and compared the effects of anti-MHVR monoclonal antibody on susceptibility of the mouse cells to MHV and RCV. The Parker (RCV-P) and sialodacryoadenitis (RCV-SDAV) strains of RCV expressed the spike glycoprotein S, but only RCV-P expressed a hemagglutinin-esterase glycoprotein that had acetylesterase activity. Therefore RCV-SDAV must bind to cellular receptors by the viral S glycoprotein, whereas RCV-P might bind to cells by its hemagglutinin-esterase glycoprotein as well as by S. Pretreatment of L2(Percy) 41.a or CMT-93 cells with anti-MHVR monoclonal antibody blocked infection with MHV-A59 but did not prevent infection of these murine cells with RCV-P or RCV-SDAV. Baby hamster kidney cells transfected with cDNAs encoding MHVR (Bgp1a) or Bgp2 were susceptible to MHV-A59 but not to RCV-P or RCV-SDAV. Thus the RCV strains cannot use these murine coronavirus receptors and must be infecting murine cells by another, as yet unknown, receptor.

Growth characteristics and protein profiles of prototype and wild-type rat coronavirus isolates grown in a cloned subline of mouse fibroblasts (L2p.176 cells).

Rat coronaviruses (RCVs) infect laboratory rats and confound biomedical research results. In vitro systems developed so far have limited the growth in knowledge about RCVs by not permitting generation of plaque-cloned virus stocks, reliable isolation of RCVs from rat tissues, or growth of high titered stocks of all isolates. Due to the fact that less than 20% of L2(Percy) cells were becoming infected, sublines were produced and selected for maximal growth of RCVs. Screening of 238 cell sublines yielded L2p.176 cells which were highly susceptible to all RCVs tested; however, susceptibility declined after 30 passages in vitro. Low-passaged L2p.176 cells were used to isolate virus from natural outbreaks and to propagate individual RCV plaques into high titered stocks. Proteins from six RCV isolates were immunoblotted using polyclonal rat and mouse antibodies to sialodacryoadenitis virus and polyclonal monospecific rabbit and goat antibodies against the peplomer (S) and nucleocapsid (N) proteins of mouse hepatitis virus (MHV). Proteins of two prototype, one Japanese and three wild type RCVs were examined and found to be similar to those of MHV, although the exact sizes and ratios of protein forms were unique for most RCV isolates. This study reports the development of a continuous cell line which reliably supports RCVs opening an opportunity for further in vivo studies of the biology of these agents. As a first step in the characterization of RCVs, we have shown that RCV proteins are very similar to those of MHV.

Comparative severity of respiratory lesions of sialodacryoadenitis virus and Sendai virus infections in LEW and F344 rats.

In several chronic diseases, lesions are more severe in LEW rats than in F344 rats. To determine whether or not acute viral diseases also are more severe in LEW rats than in F344 rats, we inoculated 6-7-week-old LEW and F344 rats with 10(7.2) cell culture infective units of sialodacryoadenitis virus or 10(4.7) infective units of Sendai virus. Twenty-four rats of each strain were given each virus. Lesions in nasal passages, tracheas, intrapulmonary airways, and pulmonary alveoli in 6 or 12 rats inoculated with each virus were assessed by scoring 5, 10, and 14 days after inoculation. Both viruses caused typical patchy necrotizing rhinitis, tracheitis, bronchitis, and bronchiolitis, with multifocal pneumonitis, in rats of both strains. Mean lesion indices for LEW rats given sialodacryoadenitis virus were significantly different from those for F344 rats for nasal passages on days 10 (0.999 vs. 0.680) and 14 (0.736 vs. 0.278), bronchi on day 5 (0.479 vs. 0.361), and alveoli on day 5 (0.677 vs. 0.275). Lesion indices for LEW rats given Sendai virus were significantly different from those for F344 rats for nasal passages on days 10 (1.000 vs. 0.611) and 14 (0.778 vs. 0.583); trachea on day 10 (0.625 vs. 0.028); bronchi on days 5 (0.476 vs. 0.331), 10 (0.123 vs. 0.013), and 14 (0.038 vs. 0); and alveoli on days 5 (0.413 vs. 0.114) and 10 (0.185 vs. 0.020). Thus, at the tested doses, both viruses caused more severe respiratory tract lesions in LEW rats than in F344 rats.

Effect of time of exposure to rat coronavirus and Mycoplasma pulmonis on respiratory tract lesions in the Wistar rat.

The effects of time of exposure on the progression of pulmonary lesions in rats inoculated with Mycoplasma pulmonis and the rat coronavirus, sialodacryoadenitis virus (SDAV) were studied, using six groups of 18 SPF Wistar rats (n = 108). Rats were inoculated intranasally as follows: Group 1, sterile medium only; Group 2, sterile medium followed one week later by 150 TCID50 SDAV; Group 3, sterile medium followed by 10(5.7) colony forming units of M. pulmonis; Group 4, SDAV followed one week later by M. pulmonis; Group 5, M. pulmonis followed one week later by SDAV; Group 6, M. pulmonis followed two weeks later by SDAV. Six rats from each group were euthanized at one, two and three weeks after the final inoculation. In a separate experiment, six additional animals were inoculated in each of groups 3, 5 and 6 (n = 18) and were sampled at five weeks after they had received M. pulmonis. Bronchoalveolar lavage and quantitative lung mycoplasma cultures were conducted on two-thirds of the rats. Histopathological examination and scoring of lesion severity were performed on all animals. Based on the prevalence and extent of histopathological lesions, bronchoalveolar lavage cell numbers, neutrophil differential cell counts and the isolation of M. pulmonis, the most severe disease occurred in the groups that received both agents. There was no significant difference in lesion severity between the groups receiving both agents other than in those examined during the acute stages of SDAV infection. Based on these results, it is evident that SDAV enhances lower respiratory tract disease in Wistar rats whether exposure occurs at one week prior to or at various intervals following M. pulmonis infections.

Morphologic changes in the nasal cavity associated with sialodacryoadenitis virus infection in the Wistar rat.

A sequential study of lesions of the nasal cavity associated with sialodacryoadenitis virus (SDAV) infection was made in the laboratory rat. Wistar rats were intranasally inoculated with approximately 10(3) TCID50 of the coronavirus SDAV. Transverse sections of four regions of the nasal cavity from inoculated and control animals were examined by light microscopy and immunohistochemistry at 2, 4, 6, 8, 10, and 14 days postinoculation (PI). Lesions were observed in the following regions of the upper respiratory tract: respiratory epithelium, transitional epithelium, olfactory epithelium, nasolacrimal duct, vomeronasal organ, and the submucosal glands of the nasal passages. In general, in structures lined by ciliated epithelial cells, there was focal to segmental necrosis with exfoliation of affected cells and polymorphonuclear cell infiltration during the acute stages, progressing to squamous metaplasia during the reparative stages. Repair in these regions was essentially complete by 14 days PI. In the olfactory epithelium and the vomeronasal organ, there was interstitial edema with necrosis and exfoliation of epithelial cells and minimal to moderate inflammatory cell response during the acute stages. Residual reparative lesions were still evident in the olfactory epithelium, the columnar epithelium and neuroepithelium of the vomeronasal organ, and the nasolacrimal duct at 14 days PI. Viral antigen was demonstrated by immunohistochemistry in all regions during the acute stages of the disease, with the exception of the vomeronasal organ. In view of these findings, infections of the respiratory tract with viruses such as SDAV could have significant effects on functions such as olfaction and chemoreception for > or = 2 weeks postexposure in this species.