Experimental Infection of Mexican Free-Tailed Bats (Tadarida brasiliensis) with SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus is thought to have originated in wild bats from Asia, and as the resulting pandemic continues into its third year, concerns have been raised that the virus will expand its host range and infect North American wildlife species, including bats. Mexican free-tailed bats (Tadarida brasiliensis) live in large colonies in the southern United States, often in urban areas and, as such, could be exposed to the virus from infected humans. We experimentally challenged wild T. brasiliensis with SARS-CoV-2 to determine the susceptibility, reservoir potential, and population impacts of infection in this species. Of 10 bats oronasally inoculated with SARS-CoV-2, 5 became infected and orally excreted moderate amounts of virus for up to 18 days postinoculation. These five subjects all seroconverted and cleared the virus before the end of the study with no obvious clinical signs of disease. We additionally found no evidence of viral transmission to uninoculated subjects. These results indicate that while T. brasiliensis are susceptible to SARS-CoV-2 infection, infection of wild populations of T. brasiliensis would not likely cause mortality. However, the transmission of SARS-CoV-2 from T. brasiliensis to or from humans, or to other animal species, is a possibility requiring further investigation to better define. IMPORTANCE As the COVID-19 pandemic has continued for 3+ years, there has been increasing concern that the SARS-CoV-2 virus will enter wildlife populations and potentially create new reservoirs where the virus could adapt to a new host and create variants. This is particularly possible with species that reside in man-made structures, in proximity to infected human populations. Mexican free-tailed bats (Tadarida brasiliensis) live in large colonies, often in urban settings and, thus, can be exposed by infected humans and potentially transmit the virus to new hosts. We experimentally challenged T. brasiliensis with SARS-CoV-2 and revealed that they are susceptible to the virus and excrete moderate amounts for up to 18 days postinoculation. This is important information for wildlife biologists, wildlife rehabilitation workers, and the general public that may contact these animals.

Experimental infection of Mexican free-tailed bats ( Tadarida brasiliensis) with SARS-CoV-2.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus originated in wild bats from Asia, and as the resulting pandemic continues into its third year, concerns have been raised that the virus will expand its host range and infect North American wildlife species, including bats. Mexican free-tailed bats ( Tadarida brasiliensis : TABR) live in large colonies in the southern United States, often in urban areas, and as such, could be exposed to the virus from infected humans. We experimentally challenged wild TABR with SARS-CoV-2 to determine the susceptibility, reservoir potential, and population impacts of infection in this species. Of nine bats oronasally inoculated with SARS-CoV-2, five became infected and orally excreted moderate amounts of virus for up to 18 days post inoculation. These five subjects all seroconverted and cleared the virus before the end of the study with no obvious clinical signs of disease. We additionally found no evidence of viral transmission to uninoculated subjects. These results indicate that while TABR are susceptible to SARS-CoV-2 infection, infection of wild populations of TABR would not likely cause mortality. However, the transmission of SARS-CoV-2 from TABR to or from humans, or to other animal species, is a distinct possibility requiring further investigation to better define.

Sciatic nerve conditioned medium depleted of pro-NGF modulates sodium currents and neurite outgrowth in PC12 cells.

Excitability and axon/dendrite specification are the most distinctive features in the establishment of neuronal polarization. Conditioned medium from rat sciatic nerve (CM) induced a neuronal-like morphology in PC12 cells. Here we show that CM neuritogenic activity is limited to the induction of dendrites in PC12 cells. However, treatment of these cells with CM in combination with a generic inhibitor for tyrosine kinase receptors (k252a) promoted the enhancement of neurite length, development of axons and induction of sodium currents. On the other hand, specific inhibition of TrkA and p75(NTR) receptors in CM-treated cells reduced the neurite length in comparison with cells treated only with CM, although the effect over the induction of sodium currents was continuously observed. These results suggested that CM had some components that, even though are able to start the morphological cell differentiation and produce short neurites (likely acting through TrkA and p75(NTR)), can restrain further neurite extension. Depletion of pro-NGF isoforms from CM produced a similar effect as the exerted by k252a, TrkA and p75(NTR) receptor inhibitors in CM-treated cells, inducing the elicitation of sodium currents. These results suggested that the effect of CM might be mediated through pro-NGF. The difference between the results obtained with the generic inhibitor for Trk receptors and the specific inhibitors for TrkA and p75(NTR) receptors in CM-treated cells, suggested that alternative pathways could be used to regulate neurite elongation, axon specification and sodium currents in PC12 cells. These findings represent important clues to improve the understanding of the initiation of neuronal polarity.

A real-time quantitative PCR comparative study between rat optic and sciatic nerves: determination of neuregulin-1 mRNA levels.

Injured axons from peripheral nervous system (PNS) possess the ability to regenerate. In contrast, regeneration of injured axons does not occur in the central nervous system (CNS) or occurs to a limited extent. Previous works have shown that rat sciatic nerve conditioned medium (CM) produced PC12 cells neuronal-like differentiation and neurite outgrowth. In the present work, we compared the expression of neuregulin-1s (NRG-1s) from rat sciatic and optic nerves as members of the PNS and CNS, respectively. Sciatic nerve CM showed a higher neurotrophic activity on PC12 cells than rat optic nerve CM. RT-PCR analysis verified the presence of all three types of NRG-1 mRNAs and their receptors in both types of nerves. Real-time quantitative PCR (QPCR) assays showed that the relative expression levels of all three types of NRG-1 mRNAs were higher in optic nerves than in sciatic nerves. Eleven-day cultured optic nerves showed an increased in NDF and SMDF when compared to freshly isolated optic nerves, whereas GGF decreased. However, 11-day-cultured sciatic nerves only showed an increase in SMDF mRNA. Western blots corroborated the differences in NRG-1 expression profile for both types of nerves and their CMs. Incubation of both CMs with the anti-pan-NRG-1 antibody showed that the neurotrophic activity of the optic nerve CM increased, whereas the sciatic nerve CM remained unchanged. These results indicated that different NRG-1 levels are expressed upon nerve degeneration and the balance between those levels and other neurotrophic factors could have an important role on nerve regeneration.

Association between HIV and other DNA viruses in vitro.

To investigate the association of human immunodeficiency virus (HIV) with various DNA viruses, including hepatitis B virus (HBV), cytomegalovirus (CMV) and Epstein-Barr virus, (EBV), simultaneous detection of HIV p24 antigen, HBV surface antigen and DNA, CMV-DNA and EBV-DNA expression was performed in phytohemagglutinin-stimulated peripheral blood mononuclear (PBMC) culture supernatants obtained from 54 individuals at risk for HIV infection. HIV expression in PBMC culture supernatants never occurred alone; expression of other viruses was always detected in the 24 samples expressing HIV antigen in vitro. Furthermore, in 16 patients expression of other viruses was detected without HIV expression, and in 14 patients none of the tested viruses were detected. These results indicate a strong association between the presence of HIV antibody and expression of DNA viruses in vitro (p = 0.0001). The coexpression of these viruses could be related to the evolution of HIV infection and AIDS.

Detection of human immunodeficiency virus type 1 by PCR before seroconversion in high-risk individuals who remain seronegative for prolonged periods.

HIV-1 seronegative patients at high risk for HIV infection were followed up. In 1990 PCR was positive for HIV DNA sequences in samples of 17 seronegative patients who continued to report for surveillance of HIV infection. There was clear evidence of seroconversion in four of these 17 seronegative patients, while in one patient an indeterminate result for HIV was repeatedly obtained in different samples. The other 12 patients continue to be seronegative without any evidence of HIV infection except the presence of provirus in peripheral blood mononuclear cells. It is important to apply the PCR technique together with tests to detect other virological and immunological markers, in order to identify seronegative carriers and thus avoid HIV transmission by them.

[Detection of HBV-DNA in peripheral blood mononuclear cells of anti-HIV-positive patients and its relation to other serological markers of HBV]. / Detección del ADN-VHB en células mononucleares de sangre periférica de pacientes anti-VIH-positivos, y su relación con otros marcadores serológicos del VHB.

HBV infection has been investigated in 47 anti-human HIV positive patients in relation to a similar group of 33 anti-HIV negative patients. Serological HBV markers were found in 87% of anti-HIV positive patients. The difference in markers of viral replication (HBeAg, HBV-DNA) was not statistically significant between the two groups. It has been suggested that HBV infection of peripheral blood mononuclear cells could be a cofactor implicated in the development of immunodeficiency due to HIV. For this reason we have investigated the presence of HBV-DNA in peripheral blood mononuclear cells by in situ hybridization. Although its detection was more frequent in anti-HIV positive patients than in anti-HIV negative ones (p < 0.05), it was not related to clinical state of immunodeficiency. With regard to serological HBV markers, HBV-DNA was detected in peripheral blood mononuclear cells from antiHBc w/o antiHBs patients. This fact means the virus may persist in this cells after recovery and suggest they could serve as additional reservoirs of HBV. These cells, that contain the HBV genome, could be implicated in the perpetuation, reactivation of the infection and in its transmission.

[Detection of hepatitis B virus DNA in peripheral blood mononuclear cells from patients with various hepatopathies using in situ hybridization]. / Detección del DNA del virus de la hepatitis B en células mononucleares de sangre periférica de pacientes con distintas hepatopatías mediante hibridación "in situ".

We have investigated, by "in situ" hibridisation, the presence of hepatitis B virus (HBV) DNA in peripheral blood mononuclear cells (PBMC) from 45 patients with acute and chronic hepatic disorders directly related with HBV or with some seric HBV marker. Results has been related with serological markers and the different types of hepatopaties. The HBV-DNA was detected in PBMC more frequently in patients with hepatic alterations more prolongated (chronic active hepatitis, chronic persistent hepatitis and cirrhosis) than in acute hepatitis patients. It was not detected in any asymptomatic patient with HBV serological markers. As regards HBV serological markers, HBV-DNA was detected in PBMC in 8/11 HBsAg positive patients and in 11/34 HBsAg negative patients: 3 antiHBc positive, 5 antiHBc and antiHBs positive and 3 without conventional seric markers. The detection of HBV-DNA in antiHBc and/or antiHBs positive subjects means the virus may persist after recovery of infection and suggests PMBC could serve as additional reservoirs for reinfection of hepatocytes leading to a reactivation of the liver disease. Our results suggest that HBV infection of PBMC is a frequent event during HBV infection and can have important consequences fundamentally with respect to pathogenic mechanisms of HBV induced liver disease and to the transmission of the virus.

[Detection and antigenic distribution of hepatitis B virus in liver tissue and its relation to other serological markers of viral replication]. / Detección y distribución antigénica del virus de la hepatitis B en tejido hepático y su relación con otros marcadores serológicos de replicación viral.

The aim of this work has been the production of specific monoclonal antibodies against HBV-antigens and their utilisation in order to study their distribution on liver tissue. The monoclonal antibodies anti-HBc and anti-HBs were obtained by the modified hybridoma technique. This study was performed on 50 patients affected by several chronic hepatopathies. For the detection of the antigens, avidin-biotin-peroxidase complex immunostaining was used. Both cytoplasmic and membranous HBsAg were detected in 15 out of 16 HBsAg+ patients; 8 of 12 HBsAg-/anti-HBc+ patients and 1 HBsAg-/antiHBc- patient. Cytoplasmic and nuclear HBcAg was observed in 12 of 16 HBsAg+ patients and 4 of 20 HBsAg- patients. Although the presence of serum HBsAg is an index of liver infection, in some HBsAg-/antiHB+ patients (20%) with undetectable levels of HBsAg, hepatic injury may be disclosed by the detection of other markers of active viral replication.

[Hepatitis B virus (HBV) replication in patients with various types of chronic hepatopathy]. / Estudio de la replicación del virus de la hepatitis B (VHB) en pacientes con distintas formas de hepatopatía crónica.

We study retrospectively the viral replication state (HBV) of 50 patients with chronic hepatic alterations. The seric DNA-HBV and/or intrahepatic (molecular hybridization), the intrahepatic distribution of HBV antigens (specific monoclonal antibodies labelled with immunoperoxidase), conventional seric HBV markers (commercial enzymoimmunoessay) and the different histopathologic features. We found a correlation between DNA-HBV "in situ" and HBcAg intrahepatic and the seric DNA-HBV production. 81% of the patients with HBsAg (+) had intrahepatic HBcAg and 85% (11/13) of them showed the antigen in their cytoplasms. Patients with HBcAg also had seric and liver DNA-HBV (+). The lack of seric HBsAg did not mean that non-active replication of HBV did not exist because 20% of the patients with HBsAg (-) showed seric and "in situ" DNA-HBV and cytoplasmic HBcAg. The detection of DNA-HBV in endothelial cells and vascular elements in hepatic tissue show that the rate of the HBV host cells is greater.

[Presence of HBsAg in lymphocyte cultured from patients infected with human immunodeficiency virus (HIV)]. / Presencia de HBsAg en cultivos de linfocitos de pacientes infectados por el virus de la inmunodeficiencia humana (VIH).

The behavior of the hepatitis B virus was investigated in mononuclear cell cultures in nine patients infected by the human immunodeficiency virus. Although only one of them was a carrier of HBsAg and four had anti-HBs in their sera, HBsAg was detected in the supernatant of the cultures from all patients. These results suggest that mononuclear cells might act as a reservoir for hepatitis B virus, and that the concomitant infection by this virus and human immunodeficiency virus may alter the natural evolution of any of both conditions.

[Detection of human immunodeficiency virus in seronegative adults]. / Detección del virus de la inmunodeficiencia humana en adultos seronegativos.

The diagnosis of human immunodeficiency virus infection is sometimes difficult or nonspecific, both in early and late stages, as the patients may be seronegative at the time of testing. Although serologic testing usually suffices to identify infected individuals and to follow up the course of the infection, in some cases direct detection of the virus is required. The culture of the mononuclear cells of the patient permits, after stimulation with mitogens and interleukin-2, the expression of viral antigens even in asymptomatic patients with latent or apparently nonproductive infection. In this way we have recovered the virus in four patients without serological evidence of infection. The possibility that human immunodeficiency virus infection can be undetectable with the usual diagnostic techniques, at least in a small proportion of patients, supports the need to use other methods such as direct viral culture to permit the identification of a greater number of infected individuals and the adoption of the appropriate prophylactic or therapeutic measures.

IgE antibody against surface antigens of Leishmania promastigotes in American cutaneous leishmaniasis.

As IgE-mediated immune mechanisms participate in the host defence against some types of parasites, we evaluated sera from American cutaneous leishmaniasis (ACL) patients for the presence of this antibody against Leishmania. Using monoclonal antibodies against human IgE and an immunoperoxidase staining technique, 48% of the patients sera tested were found to contain IgE antibody that bound strongly to Leishmania promastigotes. A much lower proportion of sera from non-symptomatic subjects from either endemic or non-endemic areas of the disease contained significant levels of anti-Leishmania IgE antibody (6.5% and 0% respectively). The results indicated that the IgE antibody bound predominantly to surface components of the promastigotes, and reactivity against the intracellular amastigote form of the parasite was rarely detected. Somewhat unexpectedly, in a small proportion of the sera, the IgE antibody showed apparent specificity for L. mexicana or L. braziliensis. This study demonstrates that ACL patients can develop anti-Leishmania IgE antibody responses, that seem to be directed preferentially against surface antigens of promastigotes, and that can be strain specific. This raises the question as to the possible contribution of this antibody to the immune defence mechanisms against the parasite.

In situ detection of amastigotes in American cutaneous leishmaniasis, using monoclonal antibodies.

Using an immunoperoxidase technique we have applied monoclonal antibodies against American Leishmania for the detection of amastigotes in biopsies from cutaneous leishmaniasis patients. The immunocytochemical procedure was notably superior to conventional histological staining in terms of the visualization and definition of the amastigotes. This technique could eventually prove to be of value in epidemiological studies, and possibly have prognostic importance, by allowing the in situ characterization of the species of infecting organism.

Incidence of atopic disease in a tropical environment: partial independence from intestinal helminthiasis.

The reported incidence of atopic disease in the tropical environment, albeit somewhat controversial, has often been very low. This has been postulated to be due to an inhibitory influence of intestinal helminthiasis, although the predominantly rural nature of the populations studied might also be an important factor to consider. We evaluated two tropical groups in Venezuela that were basically comparable, both being highly parasitized but one of which was urban and the other rural. The apparent incidence of allergic conditions in the urban group was, in fact, comparable to that in temperate countries, whereas that of the rural subjects was markedly lower. A similar difference was found in skin test positivity to common inhalant allergens, although reactivity to Ascaris extract was comparably high between the two groups, and total serum IgE and eosinophil levels were uniformly elevated. Our results suggest that the incidence of atopic disease in the topical environment may depend not only on the intensity of helminthiasis suffered but also on factors related to the urban-rural situation.