Heparan sulfate proteoglycans serve as alternative receptors for low affinity LCMV variants.

Members of the Old World Arenaviruses primarily utilize α-dystroglycan (α-DAG1) as a cellular receptor for infection. Mutations within the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) reduce or abrogate the binding affinity to α-DAG1 and thus influence viral persistence, kinetics, and cell tropism. The observation that α-DAG1 deficient cells are still highly susceptible to low affinity variants, suggests the use of an alternative receptor(s). In this study, we used a genome-wide CRISPR Cas9 knockout screen in DAG1 deficient 293T cells to identify host factors involved in α-DAG1-independent LCMV infection. By challenging cells with vesicular stomatitis virus (VSV), pseudotyped with the GP of LCMV WE HPI (VSV-GP), we identified the heparan sulfate (HS) biosynthesis pathway as an important host factor for low affinity LCMV infection. These results were confirmed by a genetic approach targeting EXTL3, a key factor in the HS biosynthesis pathway, as well as by enzymatic and chemical methods. Interestingly, a single point mutation within GP1 (S153F or Y155H) of WE HPI is sufficient for the switch from DAG1 to HS binding. Furthermore, we established a simple and reliable virus-binding assay, using directly labelled VSV-GP by intramolecular fusion of VSV-P and mWasabi, demonstrating the importance of HS for virus attachment but not entry in Burkitt lymphoma cells after reconstitution of HS expression. Collectively, our study highlights the essential role of HS for low affinity LCMV infection in contrast to their high affinity counterparts. Residual LCMV infection in double knockouts indicate the use of (a) still unknown entry receptor(s).

Interrogating Adaptive Immunity Using LCMV.

In this invited article, we explain technical aspects of the lymphocytic choriomeningitis virus (LCMV) system, providing an update of a prior contribution by Matthias von Herrath and J. Lindsay Whitton. We provide an explanation of the LCMV infection models, highlighting the importance of selecting an appropriate route and viral strain. We also describe how to quantify virus-specific immune responses, followed by an explanation of useful transgenic systems. Specifically, our article will focus on the following protocols. © 2020 Wiley Periodicals LLC. Basic Protocol 1: LCMV infection routes in mice Support Protocol 1: Preparation of LCMV stocks ASSAYS TO MEASURE LCMV TITERS Support Protocol 2: Plaque assay Support Protocol 3: Immunofluorescence focus assay (IFA) to measure LCMV titer MEASUREMENT OF T CELL AND B CELL RESPONSES TO LCMV INFECTION Basic Protocol 2: Triple tetramer staining for detection of LCMV-specific CD8 T cells Basic Protocol 3: Intracellular cytokine staining (ICS) for detection of LCMV-specific T cells Basic Protocol 4: Enumeration of direct ex vivo LCMV-specific antibody-secreting cells (ASC) Basic Protocol 5: Limiting dilution assay (LDA) for detection of LCMV-specific memory B cells Basic Protocol 6: ELISA for quantification of LCMV-specific IgG antibody Support Protocol 4: Preparation of splenic lymphocytes Support Protocol 5: Making BHK21-LCMV lysate Basic Protocol 7: Challenge models TRANSGENIC MODELS Basic Protocol 8: Transfer of P14 cells to interrogate the role of IFN-I on CD8 T cell responses Basic Protocol 9: Comparing the expansion of naïve versus memory CD4 T cells following chronic viral challenge.

Cell-to-cell transmission of lymphocytic choriomeningitis virus MX strain during persistent infection and its influence on cell migration.

Lymphocytic choriomeningitis virus (LCMV) can establish in its host a persistent infection, without any prominent symptoms. Even during this infection, when the infectious virions are not released, the virus still disseminates effectively. A very effective and fast way of infection of neighboring cells utilized by many viruses is cell-to-cell transmission. Viruses use different ways of cell-to-cell spread through the extracellular space or by intracellular means through different protrusions. We have found that LCMV strain MX may use three different types of cell-to-cell transport. Firstly, similar to vaccinia virus, it can use actin to propel the virus towards the neighboring cell. Secondly, virus can travel through the intracellular space inside the tunneling nanotubes, that connect the cells even at longer distances and thirdly, the virus may travel on the surface of the membrane of different protrusions connecting two cells. We have also proved that the cells infected by MX strain of LCMV migrate faster than the uninfected cells or cells infected with a different LCMV strain. In accordance with faster migration, the infected cells form more lamellipodia with high expression of keratin 1. In this work, we have introduced three types of cell-to-cell transmission utilized by strain MX of LCMV and showed that even if the cells are not in tight connection, the virus forces them to migrate faster to join the nearest cell. As we show in this work, the virus may use more than one strategy to move to another cell, while each strategy can substitute another. These ways of transmission are very fast and effective and may have a serious impact on the host. Moreover, targeting the cell-to-cell spread, by inhibiting for instance GTPase dynamin, could be an effective way of virus elimination. Keywords: lymphocytic choriomeningitis virus; transmission; migration; keratin 1; nucleoprotein.

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.

The impact of illegal waste sites on a transmission of zoonotic viruses.

BACKGROUND: Illegal waste disposal impacts public health and causes aesthetic and environmental pollution. Waste disposed in places without permitted and controlled facilities can provide a ready source of nutrition and shelter for rodents and thus promote the spread of their ecto- and endoparasites. The presence of two distinct zoonotic viruses, lymphocytic choriomeningitis virus (LCMV) and tick-borne encephalitis virus (TBEV), was searched at illegal waste sites. The aim of this study was to determine the prevalence of infection with both viruses in rodents and to discuss the virus-rodent relations in such environments. METHODS: Rodents sampled between October 2011 and April 2013 at 7 locations in the Istrian peninsula, were identified morphologically and genetically to minimize misidentification. Serological and molecular techniques were used to determine seroprevalence of infection in rodents and to detect viral RNAs. Serological testing was performed by immune fluorescence assay for detection of LCMV and TBEV specific antibodies. Real-time RT PCR was used for the detection of LCMV nucleoprotein gene and TBEV 3' non-coding region. Data were statistically analysed using SPSS statistic v2.0. RESULTS: Out of 82 rodent sera tested, the presence of LCMV antibodies was demonstrated in 24.93%. The highest prevalence of LCMV infection was found in commensal Mus musculus (47.37%), followed by 11.53%, 19.04% and 25% prevalence of infection in A. agrarius, A. flavicolis and A. sylvaticus, respectively. The highest prevalence of infection in rodents (53.33%) was found in locations with large waste sites and high anthropogenic influence. LCMV seroprevalence was significantly lower in rodents sampled from natural habitats. Viral nucleic acids were screened in 46 samples but yielded no amplicons of LCMV or TBEV. In addition, TBEV specific antibodies were not detected. CONCLUSIONS: Illegal waste sites have considerable impact on the area where they are located. Results have shown that the transmission of human pathogens can be significantly increased by the presence of waste sites. However, the pathogen must be endemic in the environment where the waste site is located. The introduction of a human pathogen as a consequence of the waste site in the area of interest could not be proven.

[Meningitis after a mouse bite]. / Meningitis na muizenbeet.

BACKGROUND: Infection with the lymphocytic choriomeningitis virus is a human zoonosis caused by a rodent-borne arenavirus and is often seen in autumn and winter when mice retreat into houses. Infection in humans is acquired after inhalation of aerosols or direct contact with excreta of an infected rodent. CASE DESCRIPTION: A 37-year-old woman was referred to St. Elisabeth hospital in Tilburg, Netherlands, complaining of severe progressive headache, nausea and vomiting. Three weeks before presentation a mouse had bitten her finger. On neurological examination there were no abnormalities. Cerebrospinal fluid investigations indicated viral meningitis. Immunofluorescence serological testing confirmed the diagnosis of lymphocytic choriomeningitis. CONCLUSION: Infection by lymphocytic choriomeningitis virus after contact with rodents can cause viral meningitis. The acquired form of the disease is known to be self-limiting in immunocompetent patients.

Notes from the field: a cluster of lymphocytic choriomeningitis virus infections transmitted through organ transplantation - Iowa, 2013.

On April 26, 2013, the United Network for Organ Sharing reported to CDC a cluster of ill organ transplant recipients in Iowa with a common organ donor. Infection with lymphocytic choriomeningitis virus (LCMV) was suspected. LCMV is a rodent-borne virus that most commonly causes nonfatal, influenza-like illness and occasional aseptic meningitis, but when transmitted through organ transplantation or in utero can cause severe, life-threatening disease.

Intrinsic defects in CD8 T cells with aging contribute to impaired primary antiviral responses.

Aging is associated with altered immune responses, particularly with a diminished CD8 T cell response. Although both intrinsic and extrinsic factors are hypothesized to impact this decreased T cell response, the direct evidence of an intrinsic deficiency in virus-specific CD8 T cells is limited. In this study, a TCR transgenic (Tg) P14 mouse model was utilized to compare the activation and proliferation of the Tg CD8 T cells of young and aged P14 mice upon stimulation with antigen or infection with virus. The proliferation of purified Tg CD8 T cells of aged mice was significantly lower than that of young mice when cultured in vitro with both the LCMV specific peptide and antigen presenting cells from young wild type mice. In addition, expression of the activation markers, CD69, CD25, and CD44, was delayed on Tg T cells of aged mice after stimulation. Importantly, while adoptive transfer of purified Tg CD8 T cells of young or aged mice into young wild type mice resulted in expansion of the Tg CD8 T cells of both ages after LCMV infection, the expansion of the Tg T cells from aged mice was significantly decreased compared with that of the Tg T cells from young mice. However, while the number of IFN-γ secreting Tg CD8 T cells from aged mice was significantly decreased compared to that of young mice, the percentages of Tg CD8 T cells producing IFN-γ were similar in young and aged mice, demonstrating that proliferation, but not function, of the Tg CD8 T cells of aged mice was impaired. Importantly, chronological age alone was not sufficient to predict an altered proliferative response; rather, expression of high levels of CD44 on CD8 T cells of aged mice reflected a decreased proliferative response. These results reveal that alterations intrinsic to CD8 T cells can contribute to the age-associated defects in the primary CD8 T cell response during viral infection.

Notes from the field: lymphocytic choriomeningitis virus infections in employees of a rodent breeding facility--Indiana, May-June 2012.

In late April 2012, an infectious disease physician contacted CDC regarding a patient with aseptic meningitis who worked at a rodent breeding facility in Indiana. Lymphocytic choriomeningitis virus (LCMV) infection was suspected, and LCMV-specific antibody was detected in blood and cerebrospinal fluid from the patient, confirming the diagnosis. LCMV is an arenavirus carried by the common house mouse. Persons become infected through close contact with infected rodents, through infected organ transplantation, or from mother to fetus. In immunocompetent adults, symptoms can range from mild febrile illness to meningeal symptoms (e.g., headache, stiff neck, or sensitivity to light). Congenitally infected infants can have a range of severe birth defects including hydrocephalus, chorioretinitis, blindness, and mental retardation (1). Infections in organ recipients, who are immunosuppressed, can have a case-fatality rate approaching 90% (2).

Solid organ transplant-associated lymphocytic choriomeningitis, United States, 2011.

Three clusters of organ transplant-associated lymphocytic choriomeningitis virus (LCMV) transmissions have been identified in the United States; 9 of 10 recipients died. In February 2011, we identified a fourth cluster of organ transplant-associated LCMV infections. Diabetic ketoacidosis developed in the organ donor in December 2010; she died with generalized brain edema after a short hospitalization. Both kidneys, liver, and lung were transplanted to 4 recipients; in all 4, severe posttransplant illness developed; 2 recipients died. Through multiple diagnostic methods, we identified LCMV infection in all persons, including in at least 1 sample from the donor and 4 recipients by reverse transcription PCR, and sequences of a 396-bp fragment of the large segment of the virus from all 5 persons were identical. In this cluster, all recipients developed severe illness, but 2 survived. LCMV infection should be considered as a possible cause of severe posttransplant illness.

Arenavirus nucleoproteins prevent activation of nuclear factor kappa B.

Arenaviruses include several causative agents of hemorrhagic fever (HF) disease in humans that are associated with high morbidity and significant mortality. Morbidity and lethality associated with HF arenaviruses are believed to involve the dysregulation of the host innate immune and inflammatory responses that leads to impaired development of protective and efficient immunity. The molecular mechanisms underlying this dysregulation are not completely understood, but it is suggested that viral infection leads to disruption of early host defenses and contributes to arenavirus pathogenesis in humans. We demonstrate in the accompanying paper that the prototype member in the family, lymphocytic choriomeningitis virus (LCMV), disables the host innate defense by interfering with type I interferon (IFN-I) production through inhibition of the interferon regulatory factor 3 (IRF3) activation pathway and that the viral nucleoprotein (NP) alone is responsible for this inhibitory effect (C. Pythoud, W. W. Rodrigo, G. Pasqual, S. Rothenberger, L. Martínez-Sobrido, J. C. de la Torre, and S. Kunz, J. Virol. 86:7728-7738, 2012). In this report, we show that LCMV-NP, as well as NPs encoded by representative members of both Old World (OW) and New World (NW) arenaviruses, also inhibits the nuclear translocation and transcriptional activity of the nuclear factor kappa B (NF-κB). Similar to the situation previously reported for IRF3, Tacaribe virus NP (TCRV-NP) does not inhibit NF-κB nuclear translocation and transcriptional activity to levels comparable to those seen with other members in the family. Altogether, our findings demonstrate that arenavirus infection inhibits NF-κB-dependent innate immune and inflammatory responses, possibly playing a key role in the pathogenesis and virulence of arenavirus.

Spatial and temporal dynamics of lymphocytic choriomeningitis virus in wild rodents, northern Italy.

We determined the prevalence of infection with lymphocytic choriomeningitis virus (LCMV) among small mammals in northern Italy and analyzed long-term dynamics of LCMV in a rodent population in the province of Trento. LCMV is circulating among the most widespread and common wild rodent species in this area (Apodemus flavicollis, Myodes glareolus, and Microtus arvalis); overall prevalence is 6.8%. During 2000-2006, intensive monitoring of LCMV in a population of yellow-necked mice (A. flavicollis) showed a positive correlation between prevalence of infection and rodent density. At the individual level, weight and sex appeared to correlate with antibody prevalence, which suggests that horizontal transmission of LCMV occurs principally among heavier, older males and occurs during fighting. Isolation and genetic characterization of this virus will be the crucial next steps for a better understanding of its ecology.

Brief report: Lymphocytic choriomeningitis virus transmitted through solid organ transplantation--Massachusetts, 2008.

Lymphocytic choriomeningitis virus (LCMV) is a rodent-borne arenavirus found worldwide. House mice (Mus musculus) are the natural reservoir, but LCMV also can infect other wild, pet, and laboratory rodents (e.g., rats, mice, guinea pigs, and hamsters). Humans can be infected through exposure to rodent excreta. Person-to-person transmission has occurred only through maternal-fetal transmission and solid organ transplantation. LCMV infection in humans can be asymptomatic or cause a spectrum of illness ranging from isolated fever to meningitis and encephalitis. Overall case fatality is <1%. Fetal infections can result in congenital abnormalities or death. Immunosuppressed patients, such as organ transplant recipients, can develop fatal hemorrhagic fever-like disease. Transmission of LCMV and an LCMV-like arenavirus via organ transplantation has been documented in three previous clusters. Of 11 recipients described in those clusters, 10 died of multisystem organ failure, with LCMV-associated hepatitis as a prominent feature. The surviving patient was treated with ribavirin (an antiviral with in vitro activity against LCMV) and reduction of immunosuppressive therapy. On April 15, 2008, an organ procurement organization (OPO) notified CDC of severe illness in two kidney transplant recipients from a common donor; at the time of notification, one of the recipients had died. Samples from the donor and both recipients were tested at CDC; on April 22, test results revealed evidence of acute LCMV infection in the donor and both recipients. This report summarizes the results of the subsequent public health investigation.

Mouse-to-human transmission of variant lymphocytic choriomeningitis virus.

A case of lymphocytic choriomeningitis virus (LCMV) infection led to investigation of the reservoir. LCMV was detected in mice trapped at the patient's home, and 12 isolates were recovered. Genetic analysis showed that human and mouse LCMVs were identical and that this LCMV strain was highly divergent from previously characterized LCMV.