Intra-Host SARS-CoV-2 Evolution in the Gut of Mucosally-Infected Chlorocebus aethiops (African Green Monkeys).

In recent months, several SARS-CoV-2 variants have emerged that enhance transmissibility and escape host humoral immunity. Hence, the tracking of viral evolutionary trajectories is clearly of great importance. Little is known about SARS-CoV-2 evolution in nonhuman primate models used to test vaccines and therapies and to model human disease. Viral RNA was sequenced from rectal swabs from Chlorocebus aethiops (African green monkeys) after experimental respiratory SARS-CoV-2 infection. Two distinct patterns of viral evolution were identified that were shared between all collected samples. First, mutations in the furin cleavage site that were initially present in the virus as a consequence of VeroE6 cell culture adaptation were not detected in viral RNA recovered in rectal swabs, confirming the necessity of this motif for viral infection in vivo. Three amino acid changes were also identified; ORF 1a S2103F, and spike D215G and H655Y, which were detected in rectal swabs from all sampled animals. These findings are demonstrative of intra-host SARS-CoV-2 evolution and may identify a host-adapted variant of SARS-CoV-2 that would be useful in future primate models involving SARS-CoV-2 infection.

High levels of undiagnosed rectal STIs suggest that screening remains inadequate among Black gay, bisexual and other men who have sex with men.

OBJECTIVE: To better understand rectal STI screening practices for Black gay, bisexual and other men who have sex with men (BGBMSM). FINDINGS: Although 15% of BGBMSM lab tested positive for a rectal STI, the majority of these (94%) were asymptomatic. Though all participants reported their status as HIV negative/unknown, 31 of 331 (9.4%) tested positive on HIV rapid tests. Neither condomless anal intercourse nor the number of male sex partners was associated with rectal STI or HIV diagnosis, although rectal STI diagnosis was positively related to testing HIV positive. CONCLUSIONS: Findings suggest that substantial numbers of BGBMSM have asymptomatic STIs but are not tested-an outcome that is likely a strong driver of onward HIV acquisition. Therefore, we must address the asymptomatic STI epidemic among GBMSM in order to reduce HIV transmission, as well as temper STI transmission, among this key population.

Serial Screening for SARS-CoV-2 in Rectal Swabs of Symptomatic COVID-19 Patients.

We used serial rectal swabs to investigate the amount and duration of virus secretion through the gastrointestinal tract and assessed the association between fecal shedding and gastrointestinal symptoms and to clarify the clinical usefulness testing rectal swabs. We enrolled ten adult patients hospitalized with symptomatic coronavirus disease 2019 (COVID-19). Respiratory and stool specimens were collected by physicians. The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed using real-time reverse-transcription polymerase chain reaction. All ten patients had respiratory symptoms, six had diarrhea, and seven were positive for SARS-CoV-2 on rectal swabs. The viral loads in the respiratory specimens was higher than those in the rectal specimens, and no rectal specimens were positive after the respiratory specimens became negative. There was no association between gastrointestinal symptoms, pneumonia, severity, and rectal viral load. Rectal swabs may play a role in detecting SARS-CoV-2 in individuals with suspected COVID-19, regardless of gastrointestinal symptoms.

Susceptibility of livestock to SARS-CoV-2 infection.

We report pilot studies to evaluate the susceptibility of common domestic livestock (cattle, sheep, goat, alpaca, rabbit, and horse) to intranasal infection with SARS-CoV-2. None of the infected animals shed infectious virus via nasal, oral, or faecal routes, although viral RNA was detected in several animals. Further, neutralizing antibody titres were low or non-existent one month following infection. These results suggest that domestic livestock are unlikely to contribute to SARS-CoV-2 epidemiology.

UK B.1.1.7 (Alpha) variant exhibits increased respiratory replication and shedding in nonhuman primates.

The continuing emergence of SARS-CoV-2 variants calls for regular assessment to identify differences in viral replication, shedding and associated disease. In this study, we compared African green monkeys infected intranasally with either the UK B.1.1.7 (Alpha) variant or its contemporary D614G progenitor. Both variants caused mild respiratory disease with no significant differences in clinical presentation. Significantly higher levels of viral RNA and infectious virus were found in upper and lower respiratory tract samples and tissues from B.1.1.7 infected animals. Interestingly, D614G infected animals showed significantly higher levels of viral RNA and infectious virus in rectal swabs and gastrointestinal tissues. Our results indicate that B.1.1.7 infection in African green monkeys is associated with increased respiratory replication and shedding but no disease enhancement similar to human B.1.1.7 cases.

Protective efficacy of the anti-HIV broadly neutralizing antibody PGT121 in the context of semen exposure.

BACKGROUND: HIV-1 infections occur following viral exposure at anogenital mucosal surfaces in the presence of semen. Semen contains immunosuppressive and pro-inflammatory factors. Semen from HIV-1-infected donors contains anti-HIV-1 antibodies. We assessed if passively infused anti-HIV-1 neutralizing antibody conferred protection from rectal SHIVSF162P3 challenge at semen exposed mucosae. METHODS: We pooled seminal plasma from HIV-1-infected donors. The pool was screened by ELISA for antibodies against HIV-1SF162 gp140. The ability of seminal plasma to inhibit macaque NK cells from responding to direct and antibody-dependent stimulation was assessed. The ability of seminal plasma to inhibit macaque granulocytes from mediating oxidative burst was also assessed. To demonstrate viral infectivity in the presence of seminal plasma, macaques (n = 4) were rectally challenged with SHIVSF162P3 following exposure to 2.5 mL of seminal plasma. To evaluate if anti-HIV-1 neutralizing antibody confers protection against rectal SHIV challenge at semen exposed mucosae, eight macaques were intravenously infused with PGT121, either wild type (n = 4) or the Fc receptor binding deficient LALA variant (n = 4), and rectally challenged with SHIVSF162P3 following exposure to 2.5 mL of seminal plasma. FINDINGS: Anti-HIV-1SF162 gp140 antibodies were detected in seminal plasma. Seminal plasma inhibited direct and antibody-dependent NK cell activation and granulocyte oxidative burst in vitro. Rectal SHIVSF162P3 challenge of control macaques following seminal plasma exposure resulted in infection of all animals. All macaques infused with wild type or LALA PGT121 and challenged with SHIVSF162P3 following seminal plasma exposure were protected. INTERPRETATION: PGT121 conferred protection against rectal SHIVSF162P3 challenge at semen exposed mucosae. Future research should investigate if semen alters protection conferred by antibodies more dependent on non-neutralizing functions. FUNDING: This work was supported by a grant from the Australian National Health and Medical Research Council (APP1124680).

Safety Assessment of Microbicide 2P23 on the Rectal and Vaginal Microbiota and Its Antiviral Activity on HIV Infection.

Containment of the AIDS pandemic requires reducing HIV transmission. HIV infection is initiated by the fusion of the membrane between the virus and the cell membrane of the host. 2P23 is an effective HIV membrane fusion inhibitor that may be a good entry inhibitor microbicide candidate. This study evaluated the potential of using gel-formulated 2P23 as a topical microbicide to prevent sexual transmission of HIV in the rectum and vagina. Our data revealed that 2P23 formulated in gel is effective against HIV. There was no change in antiviral activity at 25°C for 4 months or 60°C for 1 week. In addition, we demonstrated that the 2P23 gel was stable and fully functional at pH 4.0-8.0 and under different concentrations of H2O2. Finally, the 2P23 gel exhibited no cytotoxicity or antimicrobial activity and did not induce inflammatory changes in the rectal or vaginal mucosal epithelium in New Zealand rabbits after 20 mg/day daily rectovaginal application for 14 consecutive days. Despite repeated tissue sampling and 2P23 gel treatment, the inflammatory cytokines and microbiota of the rectum and vagina remained stable. These results add to general knowledge on the in vivo evaluation of anti-HIV microbicide application concerning inflammatory cytokines and microbiota changes in the rectum and vagina. These findings suggest that the 2P23 gel is an excellent candidate for further development as a safe and effective pre-exposure prophylactic microbicide for the prevention of HIV transmission.

Movements of Indian Flying Fox in Myanmar as a Guide to Human-Bat Interface Sites.

Frugivorous bats play a vital role in tropical ecosystems as pollinators and seed dispersers but are also important vectors of zoonotic diseases. Myanmar sits at the intersection of numerous bioregions and contains habitats that are important for many endangered and endemic species. This rapidly developing country also forms a connection between hotspots of emerging human diseases. We deployed Global Positioning System collars to track the movements of 10 Indian flying fox (Pteropus giganteus) in the agricultural landscapes of central Myanmar. We used clustering analysis to identify foraging sites and high-utilization areas. As part of a larger viral surveillance study in bats of Myanmar, we also collected oral and rectal swab samples from 29 bats to test for key emerging viral diseases in this colony. There were no positive results detected for our chosen viruses. We analyzed their foraging movement behavior and evaluated selected foraging sites for their potential as human-wildlife interface sites.

Comparison of SARS-CoV-2 Variants of Concern 202012/01 (U.K. Variant) and D614G Variant Transmission by Different Routes in Syrian Hamsters.

Introduction: Many SARS-CoV-2 variants of concern (VOC) have been reported recently that were linked to increased transmission. In our earlier study using VOC 202012/01 (U.K. variant) and D614G variant in the hamster model, we observed higher viral RNA shedding through nasal wash in the case of U.K. variant with lower pathogenicity in lung. In this study, we have studied transmission of these two variants by direct contact, aerosol, and fomite routes in Syrian hamsters and compared the viral load and body weight changes in hamsters exposed by both variants to understand the transmission efficiency. Methods: Nasal, throat, and rectal swabs were collected sequentially to assess viral load till 14 days. Results: Transmission could be established by direct, aerosol, and fomite contact in Syrian hamsters. Body weight loss or viral load in the contact animals exposed did not show any statistical significance. Conclusion: The study demonstrated comparable transmission of both U.K. and D614G variants of SARS-CoV-2 in Syrian hamsters in the given conditions. Provided these data, it seems that all the routes of exposure are effective leading to higher transmission.

Leukocytospermia induces intraepithelial recruitment of dendritic cells and increases SIV replication in colorectal tissue explants.

Mucosal exposure to infected semen accounts for the majority of HIV-1 transmission events, with rectal intercourse being the route with the highest estimated risk of transmission. Yet, the impact of semen inflammation on colorectal HIV-1 transmission has never been addressed. Here we use cynomolgus macaques colorectal tissue explants to explore the effect of leukocytospermia, indicative of male genital tract inflammation, on SIVmac251 infection. We show that leukocytospermic seminal plasma (LSP) has significantly higher concentration of a number of pro-inflammatory molecules compared to normal seminal plasma (NSP). In virus-exposed explants, LSP enhance SIV infection more efficiently than NSP, being the increased viral replication linked to the level of inflammatory and immunomodulatory cytokines. Moreover, LSP induce leukocyte accumulation on the apical side of the colorectal lamina propria and the recruitment of a higher number of intraepithelial dendritic cells than with NSP. These results suggest that the outcome of mucosal HIV-1 infection is influenced by the inflammatory state of the semen donor, and provide further insights into mucosal SIV/HIV-1 pathogenesis.

Unlikely SARS-CoV-2 Transmission During Vaginal Delivery.

Pregnant women display a higher risk of progression to disease and higher viral loads during infections due to their more permissive, tolerogenic immune system. However, only few studies have focused on SARS-CoV-2 intrapartum vertical transmission via vaginal secretions or faeces. The aim of this study was to investigate the presence of the virus in vaginal, rectal and blood specimens from pregnant women characterized by different COVID-19 disease severity. We enrolled 56 SARS-CoV-2-positive pregnant women, of which 46 (82%) were in the third trimester of pregnancy, 6 (10%) in the second and 4 (7%) in the first. QPCR was performed to detect the virus in vaginal and rectal swabs and in plasma samples. SARS-CoV-2 was detected in 27% of rectal swabs of pregnant women in the third trimester, while no virus particles were detected in vaginal swabs of the same patients. Furthermore, only 4% plasma samples tested positive to SARS-CoV-2. No virus was detected in newborn's nasopharyngeal swabs. Despite the low number of subjects enrolled, our data suggest that, while theoretically possible, intrapartum vaginal or orofecal SARS-CoV-2 transmission seems to be unlikely.

Th17 T Cells and Immature Dendritic Cells Are the Preferential Initial Targets after Rectal Challenge with a Simian Immunodeficiency Virus-Based Replication-Defective Dual-Reporter Vector.

Understanding the earliest events of human immunodeficiency virus (HIV) sexual transmission is critical to developing and optimizing HIV prevention strategies. To gain insights into the earliest steps of HIV rectal transmission, including cellular targets, rhesus macaques were intrarectally challenged with a single-round simian immunodeficiency virus (SIV)-based dual reporter that expresses luciferase and near-infrared fluorescent protein 670 (iRFP670) upon productive transduction. The vector was pseudotyped with the HIV-1 envelope JRFL. Regions of tissue containing foci of luminescent transduced cells were identified macroscopically using an in vivo imaging system, and individual transduced cells expressing fluorescent protein were identified and phenotyped microscopically. This system revealed that anal and rectal tissues are both susceptible to transduction 48 h after the rectal challenge. Detailed phenotypic analysis revealed that, on average, 62% of transduced cells are CCR6-positive (CCR6+) T cells-the vast majority of which express RORγT, a Th17 lineage-specific transcription factor. The second most common target cells were immature dendritic cells at 20%. These two cell types were transduced at rates that are four to five times higher than their relative abundances indicate. Our work demonstrates that Th17 T and immature dendritic cells are preferential initial targets of HIV/SIV rectal transmission. IMPORTANCE Men and women who participate in unprotected receptive anal intercourse are at high risk of acquiring HIV. While in vitro data have developed a framework for understanding HIV cell tropism, the initial target cells in the rectal mucosa have not been identified. In this study, we identify these early host cells by using an innovative rhesus macaque rectal challenge model and methodology, which we previously developed. Thus, by shedding light on these early HIV/SIV transmission events, this study provides a specific cellular target for future prevention strategies.

Rectal tissue and vaginal tissue from intravenous VRC01 recipients show protection against ex vivo HIV-1 challenge.

BackgroundVRC01, a potent, broadly neutralizing monoclonal antibody, inhibits simian-HIV infection in animal models. The HVTN 104 study assessed the safety and pharmacokinetics of VRC01 in humans. We extend the clinical evaluation to determine intravenously infused VRC01 distribution and protective function at mucosal sites of HIV-1 entry.MethodsHealthy, HIV-1-uninfected men (n = 7) and women (n = 5) receiving VRC01 every 2 months provided mucosal and serum samples once, 4-13 days after infusion. Eleven male and 8 female HIV-seronegative volunteers provided untreated control samples. VRC01 levels were measured in serum, secretions, and tissue, and HIV-1 inhibition was determined in tissue explants.ResultsMedian VRC01 levels were quantifiable in serum (96.2 µg/mL or 1.3 pg/ng protein), rectal tissue (0.11 pg/ng protein), rectal secretions (0.13 pg/ng protein), vaginal tissue (0.1 pg/ng protein), and cervical secretions (0.44 pg/ng protein) from all recipients. VRC01/IgG ratios in male serum correlated with those in paired rectal tissue (r = 0.893, P = 0.012) and rectal secretions (r = 0.9643, P = 0.003). Ex vivo HIV-1Bal26 challenge infected 4 of 21 rectal explants from VRC01 recipients versus 20 of 22 from controls (P = 0.005); HIV-1Du422.1 infected 20 of 21 rectal explants from VRC01 recipients and 12 of 12 from controls (P = 0.639). HIV-1Bal26 infected 0 of 14 vaginal explants of VRC01 recipients compared with 23 of 28 control explants (P = 0.003).ConclusionIntravenous VRC01 distributes into the female genital and male rectal mucosa and retains anti-HIV-1 functionality, inhibiting a highly neutralization-sensitive but not a highly resistant HIV-1 strain in mucosal tissue. These findings lend insight into VRC01 mucosal infiltration and provide perspective on in vivo protective efficacy.FundingNational Institute of Allergy and Infectious Diseases and Bill & Melinda Gates Foundation.

Northern pig-tailed macaques ( Macaca leonina) infected with SARS-CoV-2 show rapid viral clearance and persistent immune response.

Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), has become an unprecedented global health emergency. At present, SARS-CoV-2-infected nonhuman primates are considered the gold standard animal model for COVID-19 research. Here, we showed that northern pig-tailed macaques ( Macaca leonina, NPMs) supported SARS-CoV-2 replication. Furthermore, compared with rhesus macaques, NPMs showed rapid viral clearance in lung tissues, nose swabs, throat swabs, and rectal swabs, which may be due to higher expression of interferon (IFN)-α in lung tissue. However, the rapid viral clearance was not associated with good outcome. In the second week post infection, NPMs developed persistent or even more severe inflammation and body injury compared with rhesus macaques. These results suggest that viral clearance may have no relationship with COVID-19 progression and SARS-CoV-2-infected NPMs could be considered as a critically ill animal model in COVID-19 research.

Exploring the potential roles of some rodents in the transmission of the Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is one of the recently identified zoonotic coronaviruses. The one-hump camels are believed to play important roles in the evolution and transmission of the virus. The animal-to-animal, as well as the animal-to-human transmission in the context of MERS-CoV infection, were reported. The camels shed the virus in some of their secretions, especially the nasal tract. However, there are many aspects of the transmission cycle of the virus from animals to humans that are still not fully understood. Rodents played important roles in the transmission of many pathogens, including viruses and bacteria. They have been implicated in the evolution of many human coronaviruses, especially HCoV-OC43 and HCoV-HKU1. However, the role of rodents in the transmission of MERS-CoV still requires more exploration. To achieve this goal, we identified MERS-CoV that naturally infected dromedary camel by molecular surveillance. We captured 15 of the common rodents (rats, mice, and jerboa) sharing the habitat with these animals. We collected both oral and rectal swabs from these animals and then tested them by the commercial MERS-CoV real-time-PCR kits using two targets. Despite the detection of the viral shedding in the nasal swabs of some of the dromedary camels, none of the rodents tested positive for the virus during the tenure of this study. We concluded that these species of rodents did not harbor the virus and are most unlikely to contribute to the transmission of the MERS-CoV. However, further large-scale studies are required to confirm the potential roles of rodents in the context of the MERS-CoV transmission cycle, if any.

Neutralizing antibodies for SARS-CoV-2 in stray animals from Rio de Janeiro, Brazil.

The epidemic of coronavirus disease 2019 (COVID-19), caused by a novel Betacoronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a public health emergency worldwide. Few reports indicate that owned pets from households with at least one human resident that was diagnosed with COVID-19 can be infected by SARS-CoV-2. However, the exposure to SARS-CoV-2 of pets from households with no COVID-19 cases or stray animals remains less assessed. Using real-time reverse transcriptase polymerase chain reaction (RT-PCR) and plaque reduction neutralization test (PRNT90), we investigated the infection and previous exposure of dogs and cats to SARS-CoV-2 during the ongoing COVID-19 epidemic in Rio de Janeiro, Brazil. From June to August 2020, 96 animals were sampled, including 49 cats (40 owned and 9 stray) and 47 dogs (42 owned and 5 stray). Regarding owned pets, 75.6% (62/82) belonged to households with no COVID-19 cases. Samples included serum, and rectal and oropharyngeal swabs. All swabs were negative for SARS-CoV-2 RNA, but serum samples of a stray cat and a stray dog presented neutralizing antibodies for SARS-CoV-2, with PRNT90 titer of 80 and 40, respectively. Serological data presented here suggest that not only owned pets from households with COVID19 cases, but also stray animals are being exposed to SARS-CoV-2 during the COVID-19 pandemic.

Virological Characteristics of Hospitalized Children With SARS-CoV-2 Infection.

BACKGROUND AND OBJECTIVES: In children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, virological characteristics and correlation with disease severity have not been extensively studied. The primary objective in this study is to determine the correlation between SARS-CoV-2 viral load (VL) in infected children with age, disease severity, and underlying comorbidities. METHODS: Children <21 years, screened for SARS-CoV-2 at the time of hospitalization, who tested positive by polymerase chain reaction were included in this study. VL at different sites was determined and compared between groups. RESULTS: Of the 102 children included in this study, 44% of the cohort had asymptomatic infection, and children with >1 comorbidity were the most at risk for severe disease. VL in children with symptomatic infection was significantly higher than in children with asymptomatic infection (3.0 × 105 vs 7.2 × 103 copies per mL; P = .001). VL in the respiratory tract was significantly higher in children <1 year, compared with older children (3.3 × 107 vs 1.3 × 104 copies per mL respectively; P < .0001), despite most infants presenting with milder illness. Besides the respiratory tract, SARS-CoV-2 RNA was also detectable in samples from the gastrointestinal tract (saliva and rectum) and blood. In 13 children for whom data on duration of polymerase chain reaction positivity was available, 12 of 13 tested positive 2 weeks after initial diagnosis, and 6 of 13 continued to test positive 4 weeks after initial diagnosis. CONCLUSIONS: In hospitalized children with SARS-CoV-2, those with >1 comorbid condition experienced severe disease. SARS-CoV-2 VL in the respiratory tract is significantly higher in children with symptomatic disease and children <1 year of age.

Detection of Nipah virus in Pteropus medius in 2019 outbreak from Ernakulam district, Kerala, India.

BACKGROUND: In June 2019, Nipah virus (NiV) infection was detected in a 21-year-old male (index case) of Ernakulum, Kerala, India. This study was undertaken to determine if NiV was in circulation in Pteropus species (spp) in those areas where the index case had visit history in 1 month. METHODS: Specialized techniques were used to trap the Pteropus medius bats (random sampling) in the vicinity of the index case area. Throat and rectal swabs samples of 141 bats along with visceral organs of 92 bats were collected to detect the presence of NiV by real-time reverse transcriptase-polymerase chain reaction (qRTPCR). Serum samples of 52 bats were tested for anti-NiV Immunoglobulin (Ig) G antibodies by Enzyme-Linked Immunosorbent Assay (ELISA). The complete genome of NiV was sequenced by next-generation sequencing (NGS) from the tissues and swab samples of bats. RESULTS: One rectal swab sample and three bats visceral organs were found positive for the NiV. Interestingly, 20.68% (12/58) of Pteropus were positive for anti-NiV IgG antibodies. NiV sequences of 18,172; 17,200 and 15,100 nucleotide bps could be retrieved from three Pteropus bats. CONCLUSION: A distinct cluster of NiV sequences, with significant net-evolutionary nucleotide divergence, was obtained, suggesting the circulation of new genotype (I-India) in South India. NiV Positivity in Pteropus spp. of bats revealed that NiV is circulating in many districts of Kerala state, and active surveillance of NiV should be immediately set up to know the hotspot area for NiV infection.