A rapid and sensitive one step-SYBR green based semi quantitative real time RT-PCR for the detection of <Emphasis Type="Italic">peste des petits ruminants</Emphasis> virus in the clinical samples

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit® for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with T_m of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ～0.0001 cell culture infectious dose 50% units (TCID₅₀). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.     

Recovery of Viral RNA and Infectious Foot-and-Mouth Disease Virus from Positive Lateral-Flow Devices

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories.     

Evidence of alphaherpesvirus infections in Alaskan caribou and reindeer

Background  

The reindeer (Rangifer tarandus tarandus) industry in Alaska began with animals imported from Siberia (Russia) in the 1890's. Cervid herpes virus 2 (CvHV2) is endemic in reindeer in Scandinavia. We sought to determine if the same virus, or similar herpesviruses, were circulating in Alaskan reindeer and caribou (Rangifer tarandus granti). Serum samples from 292 reindeer were collected during annual reindeer handlings (1988-2005) near Nome, Alaska. In 2005, swab samples were collected from 40 calves from this herd, near Nome, Alaska. In 2007, ocular and nasal swab samples were collected from 30 apparently healthy reindeer calves near Wales, Alaska. Samples of plasma and white blood cells were collected from three Alaskan caribou herds, Mulchatna (n = 24), Teshekpuk (n = 34) and the Western Arctic (n = 87) in 2009.     

A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and Taq Man RT-PCR. The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50. The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg. Based on the serial dilution of the live-attenuated PPR vaccine virus, the detection limit was ~0.0001 cell culture infectious dose 50% units (TCID50). Additionally, swab materials spiked with known titre of vaccine virus were equally well detected in the assay. The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples. The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples. The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats. Therefore, the established, simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.     

Exocortis Disease: Evidence for a New Species of “Infectious” Low Molecular Weight RNA in Plants

THE anomalous properties of the infectious agent of exocortis disease in citrus (CEV), supposedly a virus, have been attributed^1,2 to infectious RNA existing as free nucleic acid. Although this model is consistent with the low sedimentation coefficient and the susceptibility to RNAase, the infectious molecule does not cosediment with either single stranded (ss) or double stranded (ds) RNA markers after equilibrium sedimentation in Cs₂SO₄. Resistance of the 8–16S moiety to inactivation by heat or diethylpyrocarbonate further suggests the presence of ds regions₃. A low molecular weight “infectious” RNA species, undetected in healthy tissue, has been implicated as the causal agent of exocortis disease.     

Correlation of clinical trachoma and infection in Aboriginal communities

Background

Trachoma is the leading infectious cause of blindness due to conjunctival infection with Chlamydia trachomatis. The presence of active trachoma and evidence of infection are poorly correlated and a strong immunologically-mediated inflammatory response means that clinical signs last much longer than infection. This population-based study in five Aboriginal communities endemic for trachoma in northern Australia compared a fine grading of clinical trachoma with diagnostic positivity and organism load.

Methods

A consensus fine grading of trachoma, based on clinical assessment and photograding, was compared to PCR, a lipopolysacharide (LPS)-based point-of-care (POC) and a 16S RNA-based nucleic acid amplification test (NAAT). Organism load was measured in PCR positive samples.

Results

A total of 1282 residents, or 85.2% of the study population, was examined. Taking the findings of both eyes, the prevalence of trachomatous inflammation-follicular (TF) in children aged 1–9 years was 25.1% (96/383) of whom 13 (13.7%) were PCR positive on the left eye. When clinical data were limited to the left eye as this was tested for PCR, the prevalence of TF decreased to 21.4% (82/383). The 301 TF negative children, 13 (4.3%) were PCR positive. The fine grading of active trachoma strongly correlated with organism load and disease severity (rs = 0.498, P = 0.0004). Overall, 53% of clinical activity (TF₁ or TF₂) and 59% of PCR positivity was found in those with disease scores less than the WHO simplified grade of TF.

Conclusion

Detailed studies of the pathogenesis, distribution and natural history of trachoma should use finer grading schemes for the more precise identification of clinical status. In low prevalence areas, the LPS-based POC test lacks the sensitivity to detect active ocular infection and nucleic acid amplification tests such as PCR or the 16S-RNA based NAAT performed better. Trachoma in the Aboriginal communities requires specific control measures.     

Detection of a novel Chlamydia species in captive spur-thighed tortoises (Testudo graeca) in southeastern Spain and proposal of Candidatus Chlamydia testudinis

The spur-thighed tortoise (Testudo graeca) is an endangered Mediterranean tortoise that lives in North Africa, Southern Europe and Southwest Asia. In the wake of recent legislation making their keeping as domestic animals illegal, many of these animals have been returned to wildlife recovery centers in Spain. In the present study, a population of such tortoises showing signs of ocular disease and nasal discharge was examined for the presence of Chlamydia spp. Cloacal, conjunctival and/or choanal swabs were collected from 58 animals. Using a real-time PCR specific for the family Chlamydiaceae, 57/58 animals tested positive in at least one sample. While only a few samples proved positive for C. pecorum, sequencing of the 16S rRNA gene revealed a sequence identical to previously published sequences from specimens of German and Polish tortoises. Whole-genome sequences obtained from two conjunctival swab samples, as well as ANIb, TETRA values and a scheme based on 9 taxonomic marker genes revealed that the strain present in the Spanish tortoises represented a new yet non-classified species, with C. pecorum being its closest relative. We propose to designate the new species Candidatus Chlamydia testudinis.     

The Perils of Pathogen Discovery: Origin of a Novel Parvovirus-Like Hybrid Genome Traced to Nucleic Acid Extraction Spin Columns

Next-generation sequencing was used for discovery and de novo assembly of a novel, highly divergent DNA virus at the interface between the Parvoviridae and Circoviridae. The virus, provisionally named parvovirus-like hybrid virus (PHV), is nearly identical by sequence to another DNA virus, NIH-CQV, previously detected in Chinese patients with seronegative (non-A-E) hepatitis. Although we initially detected PHV in a wide range of clinical samples, with all strains sharing ∼99% nucleotide and amino acid identity with each other and with NIH-CQV, the exact origin of the virus was eventually traced to contaminated silica-binding spin columns used for nucleic acid extraction. Definitive confirmation of the origin of PHV, and presumably NIH-CQV, was obtained by in-depth analyses of water eluted through contaminated spin columns. Analysis of environmental metagenome libraries detected PHV sequences in coastal marine waters of North America, suggesting that a potential association between PHV and diatoms (algae) that generate the silica matrix used in the spin columns may have resulted in inadvertent viral contamination during manufacture. The confirmation of PHV/NIH-CQV as laboratory reagent contaminants and not bona fide infectious agents of humans underscores the rigorous approach needed to establish the validity of new viral genomes discovered by next-generation sequencing.     

呼吸道病毒核酸快速检测方案的建立与应用

本研究旨在建立一套便携、准确、操作简便的呼吸道病毒核酸快速检测方案。通过实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)验证免提取的呼吸道病毒处理试剂(extraction-free respiratory virus treatment reagent,RTU)对病毒核酸处理的效果以及超快速荧光定量PCR仪(FQ-8A)对核酸扩增的效果;将RTU和FQ-8A结合构建呼吸道病毒核酸快速检测方案,通过荧光定量PCR仪中Ct值判断阳性检出率,以验证该方案检测临床样本时的准确性。结果表明,RTU与全自动核酸提取仪在提取效果上灵敏度相当;RTU在提取不同病毒类型样本时,与其他3种提取方法效果相当,但RTU提取时间少于5 min;FQ-8A检测呼吸道合胞病毒(respiratory syncytial virus,RSV)及腺病毒(adenovirus,ADV)与对照仪器ABI-7500具有良好一致性,kappa系数分别为0.938(P<0.001)和0.887(P<0.001),但FQ-8A耗时更短,扩增时间仅在0.5 h左右;RTU和FQ-8A相结合的快检方案与常规检测方案具有高度一致的检出率,其灵敏度为91.70%,特异度为100%,kappa系数为0.944(P<0.001)。总之,通过RTU与FQ-8A的结合构建了一套可在35 min内完成全部流程的呼吸道病毒核酸快速检测方案。该方案准确性高、操作简便,可为呼吸道病毒快速诊断和治疗提供重要支持。     

Seroconversion and fever are dose-dependent in a nonhuman primate model of inhalational COVID-19

While evidence exists supporting the potential for aerosol transmission of SARS-CoV-2, the infectious dose by inhalation remains unknown. In the present study, the probability of infection following inhalation of SARS-CoV-2 was dose-dependent in a nonhuman primate model of inhalational COVID-19. The median infectious dose, assessed by seroconversion, was 52 TCID₅₀ (95% CI: 23–363 TCID₅₀), and was significantly lower than the median dose for fever (256 TCID₅₀, 95% CI: 102–603 TCID₅₀), resulting in a group of animals that developed an immune response post-exposure but did not develop fever or other clinical signs of infection. In a subset of these animals, virus was detected in nasopharyngeal and/or oropharyngeal swabs, suggesting that infected animals without signs of disease are able to shed virus and may be infectious, which is consistent with reports of asymptomatic spread in human cases of COVID-19. These results suggest that differences in exposure dose may be a factor influencing disease presentation in humans, and reinforce the importance of public health measures that limit exposure dose, such as social distancing, masking, and increased ventilation. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, and, ultimately, mitigation strategies. Additionally, these data will be useful to inform dose selection in future studies examining the efficacy of therapeutics and vaccines against inhalational COVID-19, and as a baseline in healthy, young adult animals for assessment of the importance of other factors, such as age, comorbidities, and viral variant, on the infectious dose and disease presentation.     

Viral Dose and Immunosuppression Modulate the Progression of Acute BVDV-1 Infection in Calves: Evidence of Long Term Persistence after Intra-Nasal Infection

Bovine viral diarrhoea virus (BVDV) infection of cattle causes a diverse range of clinical outcomes from being asymptomatic, or a transient mild disease, to producing severe cases of acute disease leading to death. Four groups of calves were challenged with a type 1 BVDV strain, originating from a severe outbreak of BVDV in England, to study the effect of viral dose and immunosuppression on the viral replication and transmission of BVDV. Three groups received increasing amounts of virus: Group A received 10^2.55TCID₅₀/ml, group B 10^5.25TCID₅₀/ml and group C 10^6.7TCID ₅₀/ml. A fourth group (D) was inoculated with a medium dose (10^5.25TCID₅₀/ml) and concomitantly treated with dexamethasone (DMS) to assess the effects of chemically induced immunosuppression. Naïve calves were added as sentinel animals to assess virus transmission. The outcome of infection was dose dependent with animals given a higher dose developing severe disease and more pronounced viral replication. Despite virus being shed by the low-dose infection group, BVD was not transmitted to sentinel calves. Administration of dexamethasone (DMS) resulted in more severe clinical signs, prolonged viraemia and virus shedding. Using PCR techniques, viral RNA was detected in blood, several weeks after the limit of infectious virus recovery. Finally, a recently developed strand-specific RT-PCR detected negative strand viral RNA, indicative of actively replicating virus, in blood samples from convalescent animals, as late as 85 days post inoculation. This detection of long term replicating virus may indicate the way in which the virus persists and/or is reintroduced within herds.     

New Vaccine Design Based on Defective Genomes That Combines Features of Attenuated and Inactivated Vaccines

Background

New vaccine designs are needed to control diseases associated with antigenically variable RNA viruses. Foot-and-mouth disease (FMD) is a highly contagious disease of livestock that inflicts severe economic losses. Although the current whole-virus chemically inactivated vaccine has proven effective, it has led to new outbreaks of FMD because of incomplete inactivation of the virus or the escape of infectious virus from vaccine production premises. We have previously shown that serial passages of FMD virus (FMDV) C-S8c1 at high multiplicity of infection in cell culture resulted in virus populations consisting of defective genomes that are infectious by complementation (termed C-S8p260).

Principal Finding

Here we evaluate the immunogenicity of C-S8p260, first in a mouse model system to establish a proof of principle, and second, in swine, the natural host of FMDV C-S8c1. Mice were completely protected against a lethal challenge with FMDV C-S8c1, after vaccination with a single dose of C-S8p260. Pigs immunized with different C-S8p260 doses and challenged with FMDV C-S8c1 either did not develop any clinical signs or showed delayed and mild disease symptoms. C-S8p260 induced high titers of both FMDV-specific, neutralizing antibodies and activated FMDV-specific T cells in swine, that correlated with solid protection against FMDV.

Conclusions

The defective virus-based vaccine did not produce detectable levels of transmissible FMDV. Therefore, a segmented, replication-competent form of a virus, such as FMDV C-S8p260, can provide the basis of a new generation of attenuated antiviral vaccines with two safety barriers. The design can be extended to any viral pathogen that encodes trans-acting gene products, allowing complementation between replication-competent, defective forms.     

Recent approaches for control of E. coli and respiratory complex in Middle East

This study was conducted on 100 one-day-old broiler chicks to evaluate the effect of Poulvac E. coli vaccine in reduction of clinical signs and complications after concurrent infectious bronchitis virus (variant 02) and virulent E. coli O78 challenges. The birds were evaluated for clinical signs, mortality for 7?days post-infection, PM lesion score, average body weight and serological evaluation. Re-isolation and RT-PCR for the challenging infectious bronchitis virus (IBV) variant 02 were conducted thereafter. The results showed that the Poulvac E. coli at one-day old chicks in the presence of co-infection with virulent E. coli and IBV variant 02 provides better body weight gain at 35?days than the other groups. The challenge with IBV variant 02 alone in non-vaccinated birds doesn’t give any mortality; this indicated that the severity of IBV variant 02 increased by the presence of co-infection with Avian Pathogenic E. coli (APEc). The mortality percentage associated with both E. coli and IBV variant 02 infections in the none vaccinated group by Poulvac E. coli was 25% while this percentage was 10% of the vaccinated group. The Poulvac E. coli is not negatively affecting the immune response against different concurrent viral vaccines like Infectious bursal disease (IBD), and moreover, it improves the immune response against some others like Newcastle disease virus (NDV), Avian Influenza (AI) H5 and IBV.     

A Nucleic-Acid Hydrolyzing Single Chain Antibody Confers Resistance to DNA Virus Infection in HeLa Cells and C57BL/6 Mice

Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH7072) expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system.     

Experimental and Natural Infections of Goats with Severe Fever with Thrombocytopenia Syndrome Virus: Evidence for Ticks as Viral Vector

Background

Severe fever with thrombocytopenia syndrome virus (SFTSV), the causative agent for the fatal life-threatening infectious disease, severe fever with thrombocytopenia syndrome (SFTS), was first identified in the central and eastern regions of China. Although the viral RNA was detected in free-living and parasitic ticks, the vector for SFTSV remains unsettled.

Methodology/Principal Findings

Firstly, an experimental infection study in goats was conducted in a bio-safety level-2 (BSL-2) facility to investigate virus transmission between animals. The results showed that infected animals did not shed virus to the outside through respiratory or digestive tract route, and the control animals did not get infected. Then, a natural infection study was carried out in the SFTSV endemic region. A cohort of naïve goats was used as sentinel animals in the study site. A variety of daily samples including goat sera, ticks and mosquitoes were collected for viral RNA and antibody (from serum only) detection, and virus isolation. We detected viral RNA from free-living and parasitic ticks rather than mosquitoes, and from goats after ticks’ infestation. We also observed sero-conversion in all members of the animal cohort subsequently. The S segment sequences of the two recovered viral isolates from one infected goat and its parasitic ticks showed a 100% homology at the nucleic acid level.

Conclusions/Significance

In our natural infection study, close contact between goats does not appear to transmit SFTSV, however, the naïve animals were infected after ticks’ infestation and two viral isolates derived from an infected goat and its parasitic ticks shared 100% of sequence identity. These data demonstrate that the etiologic agent for goat cohort’s natural infection comes from environmental factors. Of these, ticks, especially the predominant species Haemaphysalis longicornis, probably act as vector for this pathogen. The findings in this study may help local health authorities formulate and focus preventive measures to contain this infection.     

Collaboration of Antibody and Inflammation in Clearance of Rabies Virus from the Central Nervous System

To investigate the involvement of various cellular and humoral aspects of immunity in the clearance of rabies virus from the central nervous system, (CNS), we studied the development of clinical signs and virus clearance from the CNS in knockout mice lacking either B and T cells, CD8⁺ cytotoxic T cells, B cells, alpha/beta interferon (IFN-α/β) receptors, IFN-γ receptors, or complement components C3 and C4. Following intranasal infection with the attenuated rabies virus CVS-F3, normal adult mice of different genetic backgrounds developed a transient disease characterized by loss of body weight and appetite depression which peaked at 13 days postinfection (p.i.). While these animals had completely recovered by day 21 p.i., mice lacking either B and T cells or B cells alone developed a progressive disease and succumbed to infection. Mice lacking either CD8⁺ T cells, IFN receptors, or complement components C3 and C4 showed no significant differences in the development of clinical signs by comparison with intact counterparts having the same genetic background. However, while infectious virus and viral RNA could be detected in normal control mice only until day 8 p.i., in all of the gene knockout mice studied except those lacking C3 and C4, virus infection persisted through day 21 p.i. Analysis of rabies virus-specific antibody production together with histological assessment of brain inflammation in infected animals revealed that clearance of CVS-F3 by 21 days p.i. correlated with both a strong inflammatory response in the CNS early in the infection (day 8 p.i.), and the rapid (day 10 p.i.) production of significant levels of virus-neutralizing antibody (VNA). These studies confirm that rabies VNA is an absolute requirement for clearance of an established rabies virus infection. However, for the latter to occur in a timely fashion, collaboration between VNA and inflammatory mechanisms is necessary.     

鸡传染性法氏囊病上海超强毒株NH99的分离与鉴定

通过对上海近郊某鸡场数群 10日龄左右的病鸡的临床症状、病理变化、病毒分离、纯化、动物回归、血清学检测及病毒核酸纯化、VP2基因序列的测定与分析 ,确认上海地区以发病日龄早 ,发病率、死亡率高为特征的鸡传染病为超强毒型鸡传染性法氏囊病 ,病原具有鸡传染性法氏囊病病毒超强毒 (vvIBDV)的分子特征 ,为vvIBDV。     

Isolating Influenza RNA from Clinical Samples Using Microfluidic Oil-Water Interfaces

The effective and robust separation of biomolecules of interest from patient samples is an essential step in diagnostic applications. We present a platform for the fast extraction of nucleic acids from clinical specimens utilizing paramagnetic PMPs, an oil-water interface, a small permanent magnet and a microfluidic channel to separate and purify captured nucleic acids from lysate in less than one minute, circumventing the need for multiple washing steps and greatly simplifying and expediting the purification procedure. Our device was able to isolate influenza RNA from clinical nasopharyngeal swab samples with high efficiency when compared to the Ambion® MagMAX^TM Viral RNA Isolation Kit, sufficiently separating nucleic acid analytes from PCR-inhibiting contaminants within the lysate while also critically maintaining high integrity of the viral genome. We find that this design has great potential for rapid, efficient and sensitive nucleic acid separation from patient sample.     

Further Assessment of Monkeypox Virus Infection in Gambian Pouched Rats (Cricetomys gambianus) Using In Vivo Bioluminescent Imaging

Monkeypox is a zoonosis clinically similar to smallpox in humans. Recent evidence has shown a potential risk of increased incidence in central Africa. Despite attempts to isolate the virus from wild rodents and other small mammals, no reservoir host has been identified. In 2003, Monkeypox virus (MPXV) was accidentally introduced into the U.S. via the pet trade and was associated with the Gambian pouched rat (Cricetomys gambianus). Therefore, we investigated the potential reservoir competence of the Gambian pouched rat for MPXV by utilizing a combination of in vivo and in vitro methods. We inoculated three animals by the intradermal route and three animals by the intranasal route, with one mock-infected control for each route. Bioluminescent imaging (BLI) was used to track replicating virus in infected animals and virological assays (e.g. real time PCR, cell culture) were used to determine viral load in blood, urine, ocular, nasal, oral, and rectal swabs. Intradermal inoculation resulted in clinical signs of monkeypox infection in two of three animals. One severely ill animal was euthanized and the other affected animal recovered. In contrast, intranasal inoculation resulted in subclinical infection in all three animals. All animals, regardless of apparent or inapparent infection, shed virus in oral and nasal secretions. Additionally, BLI identified viral replication in the skin without grossly visible lesions. These results suggest that Gambian pouched rats may play an important role in transmission of the virus to humans, as they are hunted for consumption and it is possible for MPXV-infected pouched rats to shed infectious virus without displaying overt clinical signs.