SARS-CoV-2 Surveillance of Wild Mice and Rats in North American Cities.

From July 2020 to June 2021, 248 wild house mice (Mus musculus), deer mice (Peromyscus maniculatus), brown rats (Rattus norvegicus), and black rats (Rattus rattus) from Texas and Washington, USA, and British Columbia, Canada, were tested for SARS-CoV-2 exposure and infection. Two brown rats and 11 house mice were positive for neutralizing antibodies using a surrogate virus neutralization test, but negative or indeterminate with the Multiplexed Fluorometric ImmunoAssay COVID-Plex, which targets full-length spike and nuclear proteins. Oro-nasopharyngeal swabs and fecal samples tested negative by RT-qPCR, with an indeterminate fecal sample in one house mouse. Continued surveillance of SARS-CoV-2 in wild rodents is warranted.

Infectivity and Shedding of Mouse Kidney Parvovirus After Oronasal Inoculation of C57BL/6, CD1, and NSG Mice.

Mouse kidney parvovirus (MKPV), the etiology of murine inclusion body nephropathy, has been identified globally in mice used for research, with an estimated prevalence of 10% in academic colonies. In immunodeficient strains, MKPV causes significant morbidity and mortality, and severe renal pathology. In contrast, in immunocompetent mice, the infection is subclinical and causes minimal pathology. We investigated viral infectivity and shedding in inbred C57BL/6NCrl (B6), outbred Crl:CD1(ICR) (CD1), and highly immunocompromised NOD. Cg - Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice. Four doses, ranging from 1.16 × 10 3 to 1.16 × 10 6 viral copies per microliter, of an MKPV inoculum were administered oronasally to 3 mice per dose per mouse type. All 3 types (B6, CD1, and NSG) had persistent infection with prolonged shedding in urine and feces. Viral copy number in the urine generally increased over time, while shedding in the feces was more variable. Among the 3 populations, CD1 mice developed viral shedding in urine earliest (4 wk after inoculation) and at higher levels (greater than 1 × 10 7 viral copies per microliter). B6 mice become viruric later (7 wk after inoculation), with lesser virus shed (1 × 10 6 viral copies per microliter or less). In CD1 and B6 mice, peak urine shedding occurred at 11 to 14 wk after inoculation, after which levels gradually declined until 35 wk after inoculation (study endpoint). In contrast, NSG mice did not become viruric until 10 wk after inoculation and continued to shed large amounts of virus (greater than 1 × 107 viral copies per microliter) in urine until the study endpoint. Two commercial immunofluorescent serologic assays failed to detect serum antibodies to MKPV nonstructural protein 1 as late as 58 wk after inoculation, whereas immunohistochemistry of infected renal tissue successfully detected anti-MKPV serum antibodies. These results increase our knowledge of the biology of MKPV and have practical application for development of effective screening programs for this pathogen.

Serendipitous Discovery of a Novel Murine Astrovirus Contaminating a Murine Helper T-cell Line and Incapable of Infecting Highly Immunodeficient Mice.

The unexpected seroconversion of sentinel mice in our facility to murine T lymphotrophic virus (MTLV) positivity led to our identification of a novel murine astrovirus that we designated murine astrovirus 2 (MuAstV-2). During our investigation, MuAstV-2 was found to be a contaminant of the T helper cell line (D10. G4.1) that was used to generate the MTLV antigen that we included in the multiplex fluorometric immunoassay (MFIA) that we used for sentinel screening. We eventually determined that cross-reactivity with the astrovirus generated a positive result in the MTLV assay. A confirmatory immunofluorometric assay (IFA) using the same MTLV-infected cell line yielded a similar result. However, the use of antigen prepared from MTLV-infected neonatal mouse thymus did not reproduce a positive result, leading us to suspect that the seroreactivity we had observed was not due to infection with MTLV. A mouse antibody production test showed that mice inoculated with naïve D10. G4.1 cells and their contact sentinels tested positive for MTLV using cell-line generated antigen, but tested negative in assays using MTLV antigen produced in mice. Metagenomic analysis was subsequently used to identify MuAstV-2 in feces from 2 sentinel mice that had recently seroconverted to MTLV. Two closely related astrovirus sequences (99.6% capsid identity) were obtained and shared 95% capsid amino acid identity with the MuAstV-2 virus sequenced from the D10. G4.1 cell line. These viruses are highly divergent from previously identified murine astroviruses, displaying <30% capsid identity, yet were closely related to murine astrovirus 2 (85% capsid identity), which had recently been isolated from feral mice in New York City. A MuAstV-2 specific PCR assay was developed and used to eradicate MuAstV-2 from the infected colony using a test and cull strategy. The newly identified MuAstV2 readily transmits to immunocompetent mouse strains by fecal-oral exposure, but fails to infect NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, which have significantly impaired adaptive and innate immune systems. Neither immunocompetent nor immunodeficient mice showed any astrovirus-associated pathology. MuAstV-2 may provide a valuable model for the study of specific aspects of astrovirus pathogenesis and virus-host interactions.

Plasma antibody profiles in non-human primate tuberculosis.

BACKGROUND: Tuberculosis (TB) in non-human primates (NHPs) is highly contagious, requiring efficient identification of animals infected with Mycobacterium tuberculosis. Tuberculin skin test is usually used but lacks desirable sensitivity/specificity and efficiency. METHODS: We aimed to develop an immunoassay for plasma antibodies against M. tuberculosis. A key challenge is that not all infected animals contain antibodies against the same M. tuberculosis antigen. Therefore, a multiplex panel of 28 antigens (Luminex(®) -Platform) was developed. RESULTS: Data revealed antibodies against eight antigens (Rv3875, Rv3875-Rv3874 fusion, Rv3874, Rv0934, Rv3881, Rv1886c, Rv2031, Rv3841) in experimentally infected (M. tuberculosis strains: Erdman and H37Rv) NHPs (rhesus and cynomolgus macaques). In a naturally acquired M. tuberculosis infection, rhesus macaques (n = 15) with lung TB pathology (n = 10) contained antibodies to five additional antigens (Rv0831, Rv2220, Rv0054, Rv1099, and Rv0129c). CONCLUSIONS: Results suggest that this user-friendly and easily implementable multiplex panel, containing 13 M. tuberculosis antigens, may provide a high-throughput alternative for NHP TB screening.

Multiplexed fluorometric immunoassay testing methodology and troubleshooting.

To ensure the quality of animal models used in biomedical research we have developed a number of diagnostic testing strategies and methods to determine if animals have been exposed to adventitious infectious agents (viruses, mycoplasma, and other fastidious microorganisms). Infections of immunocompetent animals are generally transient, yet serum antibody responses to infection often can be detected within days to weeks and persist throughout the life of the host. Serology is the primary diagnostic methodology by which laboratory animals are monitored. Historically the indirect enzyme-linked immunosorbent assay (ELISA) has been the main screening method for serosurveillance. The ELISA is performed as a singleplex, in which one microbial antigen-antibody reaction is measured per well. In comparison the MFIA is performed as a multiplexed assay. Since the microspheres come in 100 distinct color sets, as many as 100 different assays can be performed simultaneously in a single microplate well. This innovation decreases the amount of serum, reagents and disposables required for routine testing while increasing the amount of information obtained from a single test well. In addition, we are able to incorporate multiple internal control beads to verify sample and system suitability and thereby assure the accuracy of results. These include tissue control and IgG anti-test serum species immunoglobulin (αIg) coated bead sets to evaluate sample suitability. As in the ELISA and IFA, the tissue control detects non-specific binding of serum immunoglobulin. The αIg control (Serum control) confirms that serum has been added and contains a sufficient immunoglobulin concentration while the IgG control bead (System Suitability control), coated with serum species immunoglobulin, demonstrates that the labeled reagents and Luminex reader are functioning properly.

Validation of an enzyme-linked immunosorbent assay for detection of mouse parvovirus infection in laboratory mice.

PURPOSE: Parvoviruses are among the most prevalent infectious agents in mouse colonies. Infection in laboratory mice is confirmed by detection of serum antibodies to these agents, and most diagnostic tests cannot distinguish serogroup of the infecting agent. The principal objective of the research reported here was to develop and validate a sensitive, serogroup-specific diagnostic test that will distinguish between mouse parvovirus (MPV) and minute virus of mice (MVM) infection. METHODS: The MPV VP2 protein was expressed in bacteria, purified by use of metal-chelation chromatography, and used as antigen in an ELISA. More than 580 sera from uninfected mice and experimentally or naturally infected mice were screened by MPV indirect fluorescent antibody (IFA) test, then were re-tested using the MPV ELISA to define test sensitivity and specificity. An additional 3,700 sera were screened using a variety of tests, including the MPV ELISA and recombinant NS1 ELISA (rNS1 ELISA). RESULTS: Using MPV IFA test results as a benchmark, the MPV ELISA had sensitivity of 92.3% and specificity of 99.8%. In addition, the MPV ELISA detected anti-viral antibodies at a higher dilution of serum than did the IFA test, and confirmed the infecting agent as MPV or MVM. When compared directly in a commercial laboratory, the MPV ELISA had higher sensitivity (90.3% versus 65%) than and similar specificity (98.3% versus 99.6%) to the rNS1 ELISA. CONCLUSION: The MPV VP2 ELISA provides a sensitive, serogroup-specific alternative for diagnosis and classification of parvovirus infection in laboratory mice.