Reverse genetics systems for contemporary isolates of respiratory syncytial virus enable rapid evaluation of antibody escape mutants.

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infection in children under 5 y of age. In the absence of a safe and effective vaccine and with limited options for therapeutic interventions, uncontrolled epidemics of RSV occur annually worldwide. Existing RSV reverse genetics systems have been predominantly based on older laboratory-adapted strains such as A2 or Long. These strains are not representative of currently circulating genotypes and have a convoluted passage history, complicating their use in studies on molecular determinants of viral pathogenesis and intervention strategies. In this study, we have generated reverse genetics systems for clinical isolates of RSV-A (ON1, 0594 strain) and RSV-B (BA9, 9671 strain) in which the full-length complementary DNA (cDNA) copy of the viral antigenome is cloned into a bacterial artificial chromosome (BAC). Additional recombinant (r) RSVs were rescued expressing enhanced green fluorescent protein (EGFP), mScarlet, or NanoLuc luciferase from an additional transcription unit inserted between the P and M genes. Mutations in antigenic site II of the F protein conferring escape from palivizumab neutralization (K272E, K272Q, S275L) were investigated using quantitative cell-fusion assays and rRSVs via the use of BAC recombineering protocols. These mutations enabled RSV-A and -B to escape palivizumab neutralization but had differential impacts on cell-to-cell fusion, as the S275L mutation resulted in an almost-complete ablation of syncytium formation. These reverse genetics systems will facilitate future cross-validation efficacy studies of novel RSV therapeutic intervention strategies and investigations into viral and host factors necessary for virus entry and cell-to-cell spread.

Respiratory syncytial virus infection in the modern era.

PURPOSE OF REVIEW: Respiratory syncytial virus (RSV) continues to be a major cause of severe lower respiratory tract infection in infants, young children, and older adults. In this review, changes in the epidemiology of RSV during the coronavirus disease 2019 (COVID-19) pandemic are highlighted together with the role which increased molecular surveillance efforts will have in future in assessing the efficacy of vaccines and therapeutics. RECENT FINDINGS: The introduction of nonpharmaceutical intervention (NPIs) strategies during the COVID-19 pandemic between 2020 and 2022 resulted in worldwide disruption to the epidemiology of RSV infections, especially with respect to the timing and peak case rate of annual epidemics. Increased use of whole genome sequencing along with efforts to better standardize the nomenclature of RSV strains and discrimination of RSV genotypes will support increased monitoring of relevant antigenic sites in the viral glycoproteins. Several RSV vaccine candidates based on subunit, viral vectors, nucleic acid, or live attenuated virus strategies have shown efficacy in Phase 2 or 3 clinical trials with vaccines using RSVpreF protein currently the closest to approval and use in high-risk populations. Finally, the recent approval and future use of the extended half-life human monoclonal antibody Nirsevimab will also help to alleviate the morbidity and mortality burden caused by annual epidemics of RSV infections. SUMMARY: The ongoing expansion and wider coordination of RSV molecular surveillance efforts via whole genome sequencing will be crucial for future monitoring of the efficacy of a new generation of vaccines and therapeutics.

Development and Validation of a Pan-Genotypic Real-Time Quantitative Reverse Transcription-PCR Assay To Detect Canine Distemper Virus and Phocine Distemper Virus in Domestic Animals and Wildlife.

Canine distemper virus (CDV) is an animal morbillivirus belonging to the family Paramyxoviridae and has caused major epizootics with high mortality levels in susceptible wildlife species. In recent years, the documented genetic diversity of CDV has expanded, with new genotypes identified in India, the Caspian Sea, and North America. However, no quantitative real-time PCR (RT-qPCR) that has been validated for the detection of all genotypes of CDV is currently available. We have therefore established and characterized a pan-genotypic probe-based RT-qPCR assay based on the detection of a conserved region of the phosphoprotein (P) gene of CDV. This assay has been validated using virus strains representative of six genotypes of CDV in different sample types, including frozen tissue, formalin-fixed paraffin-embedded tissue sections, and virus isolates. The primers and probe target sequences were sufficiently conserved to also enable detection of the phocine distemper virus strains responsible for epizootics in harbor seals in the North Sea in 1988 and 2002. Comparison with two recently published RT-qPCR assays for CDV showed that under equivalent conditions the primers and probe set reported in this study were more sensitive in detecting nucleic acids from an Asia-4 genotype, which displays sequence variation in primer and probe binding sites. In summary, this validated new pan-genotypic RT-qPCR assay will facilitate screening of suspected distemper cases caused by novel genotypes for which full genome sequences are unavailable and have utility in detecting multiple CDV strains in geographical regions where multiple genotypes cocirculate in wildlife.

Molecular characterization of a bovine adenovirus type 7 (Bovine Atadenovirus F) strain isolated from a systemically infected calf in Germany.

Bovine adenovirus 7 (BAdV-7) is an unclassified member of the genus Atadenovirus with a worldwide distribution and has been reported to induce clinical disease of varying severity in infected cattle, ranging from asymptomatic infections to severe enteric or respiratory disease. In this study, we used next-generation sequencing to obtain the first complete genome sequence of a European strain of BadV-7, from pooled spleen and liver tissue obtained from a deceased newborn Limousin calf. Histopathological analysis and electron microscopy showing systemic lesions in multiple organs with intranuclear amphophilic inclusions observed in endothelial cells in multiple peripheral tissues. Virus isolation was readily achieved from tissue homogenate using bovine esophagus cells (KOP-R), a strategy which should facilitate future in vitro or in vivo BAdV-7 studies. Phylogenetic analysis of available genome sequences of BAdV-7 showed that the newly identified strain groups most closely with a recent BAdV-7 strain, SD18-74, from the USA, confirming that this newly identified strain is a member of the Atadenovirus genus. The fiber gene was found to be highly conserved within BAdV-7 strains but was highly divergent in comparison to Ovine adenovirus 7 (OAdV-7) (39.56% aa sequence identity). Furthermore, we report a variable region of multiple tandem repeats between the coding regions of E4.1 and RH5 genes. In summary, the presented pathological and molecular characterization of this case suggests that further research into the worldwide molecular epidemiology and disease burden of BAdV-7 is warranted.

Network meta-analysis correlates with analysis of merged independent transcriptome expression data.

BACKGROUND: Using meta-analysis, high-dimensional transcriptome expression data from public repositories can be merged to make group comparisons that have not been considered in the original studies. Merging of high-dimensional expression data can, however, implicate batch effects that are sometimes difficult to be removed. Removing batch effects becomes even more difficult when expression data was taken using different technologies in the individual studies (e.g. merging of microarray and RNA-seq data). Network meta-analysis has so far not been considered to make indirect comparisons in transcriptome expression data, when data merging appears to yield biased results. RESULTS: We demonstrate in a simulation study that the results from analyzing merged data sets and the results from network meta-analysis are highly correlated in simple study networks. In the case that an edge in the network is supported by multiple independent studies, network meta-analysis produces fold changes that are closer to the simulated ones than those obtained from analyzing merged data sets. Finally, we also demonstrate the practicability of network meta-analysis on a real-world data example from neuroinfection research. CONCLUSIONS: Network meta-analysis is a useful means to make new inferences when combining multiple independent studies of molecular, high-throughput expression data. This method is especially advantageous when batch effects between studies are hard to get removed.

Delineating morbillivirus entry, dissemination and airborne transmission by studying in vivo competition of multicolor canine distemper viruses in ferrets.

Identification of cellular receptors and characterization of viral tropism in animal models have vastly improved our understanding of morbillivirus pathogenesis. However, specific aspects of viral entry, dissemination and transmission remain difficult to recapitulate in animal models. Here, we used three virologically identical but phenotypically distinct recombinant (r) canine distemper viruses (CDV) expressing different fluorescent reporter proteins for in vivo competition and airborne transmission studies in ferrets (Mustela putorius furo). Six donor ferrets simultaneously received three rCDVs expressing green, red or blue fluorescent proteins via conjunctival (ocular, Oc), intra-nasal (IN) or intra-tracheal (IT) inoculation. Two days post-inoculation sentinel ferrets were placed in physically separated adjacent cages to assess airborne transmission. All donor ferrets developed lymphopenia, fever and lethargy, showed progressively increasing systemic viral loads and were euthanized 14 to 16 days post-inoculation. Systemic replication of virus inoculated via the Oc, IN and IT routes was detected in 2/6, 5/6 and 6/6 ferrets, respectively. In five donor ferrets the IT delivered virus dominated, although replication of two or three different viruses was detected in 5/6 animals. Single lymphocytes expressing multiple fluorescent proteins were abundant in peripheral blood and lymphoid tissues, demonstrating the occurrence of double and triple virus infections. Transmission occurred efficiently and all recipient ferrets showed evidence of infection between 18 and 22 days post-inoculation of the donor ferrets. In all cases, airborne transmission resulted in replication of a single-colored virus, which was the dominant virus in the donor ferret. This study demonstrates that morbilliviruses can use multiple entry routes in parallel, and co-infection of cells during viral dissemination in the host is common. Airborne transmission was efficient, although transmission of viruses expressing a single color suggested a bottleneck event. The identity of the transmitted virus was not determined by the site of inoculation but by the viral dominance during dissemination.

First detection and genetic characterization of peste des petits ruminants virus from dorcas gazelles "Gazella dorcas" in the Sudan, 2016-2017.

In May 2017, many free-ranging dorcas gazelles (Gazella dorcas) with suspected signs of peste des petits ruminants (PPR) were reported in Dinder National Park, South-Eastern Sudan. Peste des petits ruminants virus (PPRV) antigen and nucleic acid were detected in specimens from these gazelles using an immunocapture ELISA and a reverse transcription polymerase chain reaction (RT-PCR) assays. PPRV was also detected in four healthy semi-captive dorcas gazelles from two areas of Khartoum State. Phylogenetic analysis showed that these PPRV strains belonged to the lineage IV genotype. The present study demonstrates that gazelles are a potential wild small ruminant host for PPRV and may influence the epidemiology of PPR in the Sudan.

Association of Batai Virus Infection and Encephalitis in Harbor Seals, Germany, 2016.

We isolated Batai virus from the brain of a euthanized, 26-year-old, captive harbor seal with meningoencephalomyelitis in Germany. We provide evidence that this orthobunyavirus can naturally infect the central nervous system of a mammal. The full-genome sequence showed differences from a previously reported virus isolate from a mosquito in Germany.

Canine Bocavirus Type 2 Infection Associated With Intestinal Lesions.

Bocaviruses are small nonenveloped DNA viruses belonging to the Bocaparvovirus genus of the Parvoviridae family and have been linked to both respiratory and enteric disease in humans and animals. To date, 3 bocaviruses, canine bocaviruses 1 to 3 (CBoV-1-3), have been shown to affect dogs with different disease manifestations reported for infected animals. We used next-generation sequencing to identify a novel strain of canine CBoV-2 (CBoV TH-2016) in a litter of puppies that died in Thailand from acute dyspnea and hemoptysis, for which no causal pathogen could be identified in routine assays. Analysis of the complete coding sequences of CBoV TH-2016 showed that this virus was most closely related to a strain previously identified in South Korea (isolate 14D193), with evidence of genetic recombination in the VP2 gene with related strains from South Korea and Hong Kong. Use of quantitative polymerase chain reaction showed the presence of CBoV TH-2016 in several tissues, suggesting hematogenous virus spread, while only intestinal tissue was found to be positive by in situ hybridization and electron microscopy. Histologic small intestinal lesions associated with CBoV TH-2016 infection were eosinophilic intranuclear inclusion bodies within villous enterocytes without villous atrophy or fusion, similar to those previously considered pathognomonic for CBoV-1 infection. Therefore, this study provides novel insights in the pathogenicity of canine bocavirus infections and suggests that a novel recombinant CBoV-2 may result in atypical findings of CBoV infection. Although the specific cause of death of these puppies remained undetermined, a contributory role of enteric CBoV TH-2016 infection is possible.

New Avian Hepadnavirus in Palaeognathous Bird, Germany.

In 2015, we identified an avian hepatitis B virus associated with hepatitis in a group of captive elegant-crested tinamous (Eudromia elegans) in Germany. The full-length genome of this virus shares <76% sequence identity with other avihepadnaviruses. The virus may therefore be considered a new extant avian hepadnavirus.

Recombinant subgroup B human respiratory syncytial virus expressing enhanced green fluorescent protein efficiently replicates in primary human cells and is virulent in cotton rats.

UNLABELLED: Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies. IMPORTANCE: Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

Live-attenuated measles virus vaccine targets dendritic cells and macrophages in muscle of nonhuman primates.

UNLABELLED: Although live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replication in vivo are still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston-Zagreb (EZ), allowing recovery of recombinant (r)MV(EZ). Three recombinant viruses were generated that contained the open reading frame encoding enhanced green fluorescent protein (EGFP) within an additional transcriptional unit (ATU) at various positions within the genome. rMV(EZ)EGFP(1), rMV(EZ)EGFP(3), and rMV(EZ)EGFP(6) contained the ATU upstream of the N gene, following the P gene, and following the H gene, respectively. The viruses were compared in vitro by growth curves, which indicated that rMV(EZ)EGFP(1) was overattenuated. Intratracheal infection of cynomolgus macaques with these recombinant viruses revealed differences in immunogenicity. rMV(EZ)EGFP(1) and rMV(EZ)EGFP(6) did not induce satisfactory serum antibody responses, whereas both in vitro and in vivo rMV(EZ)EGFP(3) was functionally equivalent to the commercial MV(EZ)-containing vaccine. Intramuscular vaccination of macaques with rMV(EZ)EGFP(3) resulted in the identification of EGFP(+) cells in the muscle at days 3, 5, and 7 postvaccination. Phenotypic characterization of these cells demonstrated that muscle cells were not infected and that dendritic cells and macrophages were the predominant target cells of live-attenuated MV. IMPORTANCE: Even though MV strain Edmonston-Zagreb has long been used as a live-attenuated vaccine (LAV) to protect against measles, nothing is known about the primary cells in which the virus replicates in vivo. This is vital information given the push to move toward needle-free routes of vaccination, since vaccine virus replication is essential for vaccination efficacy. We have generated a number of recombinant MV strains expressing enhanced green fluorescent protein. The virus that best mimicked the nonrecombinant vaccine virus was formulated according to protocols for production of commercial vaccine virus batches, and was subsequently used to assess viral tropism in nonhuman primates. The virus primarily replicated in professional antigen-presenting cells, which may explain why this LAV is so immunogenic and efficacious.

Neurotropic virus infections as the cause of immediate and delayed neuropathology.

A wide range of viruses from different virus families in different geographical areas, may cause immediate or delayed neuropathological changes and neurological manifestations in humans and animals. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the central nervous system, frequently leaving the patient or affected animal with a poor or fatal prognosis. Mechanisms that govern neuropathogenesis and immunopathogenesis of viral infections are highlighted, using examples of well-studied virus infections that are associated with these alterations in different populations throughout the world. A better understanding of the molecular, epidemiological and biological characteristics of these infections and in particular of mechanisms that underlie their clinical manifestations may be expected to provide tools for the development of more effective intervention strategies and treatment regimens.

Pathological consequences of systemic measles virus infection.

The identification of poliovirus receptor-like 4 (PVRL4) as the second natural receptor for measles virus (MV) has closed a major gap in our understanding of measles pathogenesis, and explains how this predominantly lymphotropic virus breaks through epithelial barriers to transmit to a susceptible host. Advances in the development of wild-type, recombinant MVs which express fluorescent proteins making infected cells readily detectable in living tissues and animals, has also increased our understanding of this important and highly transmissible human disease. Thus, it is timely to review how these advances have provided new insights into MV infection of immune, epithelial and neural cells. This demands access to primate samples that help us understand the early and acute stages of the disease, which are challenging to dissect due to the mild/self-limiting nature of the infection. It also requires well-characterized and rather rare human tissue samples from patients who succumb to neurological sequelae to help study the consequences of the long-term persistence of this RNA virus in vivo. Collectively, these studies have provided unique insights into how the use of two cellular receptors, CD150 and PVRL4, governs the in vivo tissue-specific temporal patterns of virus spread and resulting pathological lesions. Analysis of tissue samples has also demonstrated the importance of differing mechanisms of virus cell-to-cell spread within lymphoid, epithelial and neural tissues in the dissemination of MV during acute and long-term persistent infections. Given the incentive to eradicate MV globally, and the inevitable question as to whether or not vaccination should cease in light of the existence of closely related morbilliviruses, a thorough understanding of measles pathological lesions is essential.

Measles vaccination of nonhuman primates provides partial protection against infection with canine distemper virus.

UNLABELLED: Measles virus (MV) is being considered for global eradication, which would likely reduce compliance with MV vaccination. As a result, children will grow up without MV-specific immunity, creating a potential niche for closely related animal morbilliviruses such as canine distemper virus (CDV). Natural CDV infection causing clinical signs has never been reported in humans, but recent outbreaks in captive macaques have shown that CDV can cause disease in primates. We studied the virulence and tropism of recombinant CDV expressing enhanced green fluorescent protein in naive and measles-vaccinated cynomolgus macaques. In naive animals CDV caused viremia and fever and predominantly infected CD150(+) lymphocytes and dendritic cells. Virus was reisolated from the upper and lower respiratory tracts, but infection of epithelial or neuronal cells was not detectable at the time points examined, and the infections were self-limiting. This demonstrates that CDV readily infects nonhuman primates but suggests that additional mutations are necessary to achieve full virulence in nonnatural hosts. Partial protection against CDV was observed in measles-vaccinated macaques, as demonstrated by accelerated control of virus replication and limited shedding from the upper respiratory tract. While neither CDV infection nor MV vaccination induced detectable cross-reactive neutralizing antibodies, MV-specific neutralizing antibody levels of MV-vaccinated macaques were boosted by CDV challenge infection, suggesting that cross-reactive VN epitopes exist. Rapid increases in white blood cell counts in MV-vaccinated macaques following CDV challenge suggested that cross-reactive cellular immune responses were also present. This study demonstrates that zoonotic morbillivirus infections can be controlled by measles vaccination. IMPORTANCE: Throughout history viral zoonoses have had a substantial impact on human health. Given the drive toward global eradication of measles, it is essential to understand the zoonotic potential of animal morbilliviruses. Morbilliviruses are thought to have evolved from a common ancestral virus that jumped species and adapted to new hosts. Recently, canine distemper virus (CDV), a morbillivirus normally restricted to carnivores, caused disease outbreaks in nonhuman primates. Here, we report that experimental CDV infection of monkeys resulted in fever and leukopenia. The virus replicated to high levels in lymphocytes but did not spread to epithelial cells or the central nervous system. Importantly, like measles virus in macaques, the infections were self-limiting. In measles-vaccinated macaques CDV was cleared more rapidly, resulting in limited virus shedding from the upper respiratory tract. These studies demonstrate that although CDV can readily infect primates, measles immunity is protective, and CDV infection is self-limiting.

Measles virus infection of epithelial cells in the macaque upper respiratory tract is mediated by subepithelial immune cells.

Measles virus (MV), one of the most contagious viruses infecting humans, causes a systemic infection leading to fever, immune suppression, and a characteristic maculopapular rash. However, the specific mechanism or mechanisms responsible for the spread of MV into the respiratory epithelium in the late stages of the disease are unknown. Here we show the crucial role of PVRL4 in mediating the spread of MV from immune to epithelial cells by generating a PVRL4 "blind" recombinant wild-type MV and developing a novel in vitro coculture model of B cells with primary differentiated normal human bronchial epithelial cells. We utilized the macaque model of measles to analyze virus distribution in the respiratory tract prior to and at the peak of MV replication. Expression of PVRL4 was widespread in both the lower and upper respiratory tract (URT) of macaques, indicating MV transmission can be facilitated by more than only epithelial cells of the trachea. Analysis of tissues collected at early time points after experimental MV infection demonstrated the presence of MV-infected lymphoid and myeloid cells contacting respiratory tract epithelium in the absence of infected epithelial cells, suggesting that these immune cells seed the infection in vivo. Thereafter, lateral cell-to-cell spread of MV led to the formation of large foci of infected cells in the trachea and high levels of MV infection in the URT, particularly in the nasal cavity. These novel findings have important implications for our understanding of the high transmissibility of measles.

Synthesis and biological evaluation of cleistocaltone A, an inhibitor of respiratory syncytial virus (RSV).

The first chemical synthesis of the phloroglucinol meroterpenoid cleistocaltone A (1) is presented. This compound, previously isolated from Cleistocalyx operculatus was reported to show promising antiviral properties. Based on a modified biosynthesis proposal, a synthetic strategy was devised featuring an intramolecular Diels-Alder reaction and an epoxidation/elimination sequence to generate the allyl alcohol handle in the side chain. The strategy was successfully executed and synthetic cleistcaltone A was evaluated against a contemporary RSV-A strain.

Concurrent Detection of a Papillomatous Lesion and Sequence Reads Corresponding to a Member of the Family Adintoviridae in a Bell's Hinge-Back Tortoise (Kinixys belliana).

An adult male Bell's hinge-back tortoise (Kinixys belliana) was admitted to a veterinary clinic due to a swelling in the oral cavity. Physical examination revealed an approximately 2.5 × 1.5 cm sized, irregularly shaped tissue mass with villiform projections extending from its surface located in the oropharyngeal cavity. An initial biopsy was performed, and the lesion was diagnosed as squamous papilloma. Swabs taken for virological examination tested negative with specific PCRs for papillomavirus and herpesvirus. Further analysis of the oropharyngeal mass via metagenomic sequencing revealed sequence reads corresponding to a member of the family Adintoviridae. The tissue mass was removed one week after the initial examination. The oral cavity remained unsuspicious in follow-up examinations performed after one, five and twenty weeks. However, a regrowth of the tissue was determined 23 months after the initial presentation. The resampled biopsy tested negative for sequence reads of Adintoviridae. Conclusively, this report presents the diagnostic testing and therapy of an oral cavity lesion of unknown origin. The significance of concurrent metagenomic determination of adintovirus sequence reads within the tissue lesion is discussed.

Infection of lymphoid tissues in the macaque upper respiratory tract contributes to the emergence of transmissible measles virus.

Measles virus (MV), a member of the family Paramyxoviridae, remains a major cause of morbidity and mortality in the developing world. MV is spread by aerosols but the mechanism(s) responsible for the high transmissibility of MV are largely unknown. We previously infected macaques with enhanced green fluorescent protein-expressing recombinant MV and euthanized them at a range of time points. In this study a comprehensive pathological analysis has been performed of tissues from the respiratory tract around the peak of virus replication. Isolation of virus from nose and throat swab samples showed that high levels of both cell-associated and cell-free virus were present in the upper respiratory tract. Analysis of tissue sections from lung and primary bronchus revealed localized infection of epithelial cells, concomitant infiltration of MV-infected immune cells into the epithelium and localized shedding of cells or cell debris into the lumen. While high numbers of MV-infected cells were present in the tongue, these were largely encapsulated by intact keratinocyte cell layers that likely limit virus transmission. In contrast, the integrity of tonsillar and adenoidal epithelia was disrupted with high numbers of MV-infected epithelial cells and infiltrating immune cells present throughout epithelial cell layers. Disruption was associated with large numbers of MV-infected cells or cell debris 'spilling' from epithelia into the respiratory tract. The coughing and sneezing response induced by disruption of the ciliated epithelium, leading to the expulsion of MV-infected cells, cell debris and cell-free virus, contributes to the highly infectious nature of MV.

Early target cells of measles virus after aerosol infection of non-human primates.

Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMV(KS)EGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., n = 3 per time point) and infected (EGFP(+)) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP(+) cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.