GP38 as a vaccine target for Crimean-Congo hemorrhagic fever virus.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is a tick-borne virus that causes severe hemorrhagic disease in humans. There is a great need for effective vaccines and therapeutics against CCHFV for humans, as none are currently internationally approved. Recently, a monoclonal antibody against the GP38 glycoprotein protected mice against lethal CCHFV challenge. To show that GP38 is required and sufficient for protection against CCHFV, we used three inactivated rhabdoviral-based CCHFV-M vaccines, with or without GP38 in the presence or absence of the other CCHFV glycoproteins. All three vaccines elicited strong antibody responses against the respective CCHFV glycoproteins. However, only vaccines containing GP38 showed protection against CCHFV challenge in mice; vaccines without GP38 were not protective. The results of this study establish the need for GP38 in vaccines targeting CCHFV-M and demonstrate the efficacy of a CCHFV vaccine candidate based on an established vector platform.

Ocozocoautla de espinosa virus and hemorrhagic fever, Mexico.

Arenavirus RNA was isolated from Mexican deer mice (Peromyscus mexicanus) captured near the site of a 1967 epidemic of hemorrhagic fever in southern Mexico. Analyses of nucleotide and amino acid sequence data indicated that the deer mice were infected with a novel Tacaribe serocomplex virus (proposed name Ocozocoautla de Espinosa virus), which is phylogenetically closely related to Tacaribe serocomplex viruses that cause hemorrhagic fever in humans in South America.

Geographic distribution of hantaviruses associated with neotomine and sigmodontine rodents, Mexico.

To increase our knowledge of the geographic distribution of hantaviruses associated with neotomine or sigmodontine rodents in Mexico, we tested 876 cricetid rodents captured in 18 Mexican states (representing at least 44 species in the subfamily Neotominae and 10 species in the subfamily Sigmodontinae) for anti-hantavirus IgG. We found antibodies against hantavirus in 35 (4.0%) rodents. Nucleotide sequence data from 5 antibody-positive rodents indicated that Sin Nombre virus (the major cause of hantavirus pulmonary syndrome [HPS] in the United States) is enzootic in the Mexican states of Nuevo León, San Luis Potosí, Tamaulipas, and Veracruz. However, HPS has not been reported from these states, which suggests that in northeastern Mexico, HPS has been confused with other rapidly progressive, life-threatening respiratory diseases. Analyses of nucleotide sequence data from 19 other antibody-positive rodents indicated that El Moro Canyon virus and Limestone Canyon virus are geographically widely distributed in Mexico.

Reevaluation of the phylogenetic relationships among Neotomini rodents (Hodomys, Neotoma, and Xenomys) and comments on the woodrat classification.

The woodrats or packrats of the genus Neotoma have been the subject of a wide array of research including paleoecology, physiology, morphological evolution, systematics, speciation, and hybridization. In recent years, much work has been done to elucidate evolutionary relationships within and between closely related species of the genus; in particular the addition of newly collected specimens from critical geographic regions has provided new opportunities for taxonomic assessment. Given these new data and their potential, parsimony (PARS), maximum likelihood (ML), and Bayesian inference (BI) analyses were conducted on DNA sequences obtained from nine individual genes (four mitochondrial loci: 12S, 16S, CoII, and Cytb; five nuclear loci: AdhI2, BfibI7, En2, Mlr, and Myh6) to estimate the phylogenetic relationships among 23 species of Neotoma. Results of these analyses depicted a wide array of phylogenetic relationships among taxa; with substantial nodal support recovered in both the ML and PARS analyses at some mid-level and terminal positions. Several individual genes, particularly 12S, AdhI2, BfibI7, CoII, and Cytb, provided support at several basal positions; however, phylogenetic resolution was limited in the other genes. A final BI analysis where the nine genes were concatenated into a single data set produced several supported clades that corresponded to previously recognized species groups (floridana, micropus, mexicana, and lepida) and the subgenus Homodontomys. Levels of genetic divergence for within-species comparisons (estimated from the Cytb data set) ranged from 0.88% (N. magister) to 6.82% (N. fuscipes); for between sister species comparisons ranged from 4.68% (N. devia and N. lepida) to 12.70% (N. angustapalata and N. nelsoni); and for members within closely related clades ranged from 8.70% (N. bryanti and N. lepida) to 12.57% (N. goldmani and N. magister). Evaluations of generic, subgeneric, and species group boundaries were explored using phylogenetic principles on the DNA sequence data presented herein, as well as morphological findings from previous studies. Results obtained suggest that the most conservative taxonomic interpretation involves the abandonment of subgeneric delineations and relies on the recognition of eight species groups (cinerea, floridana, fuscipes, lepida, mexicana, micropus, phenax, and stephensi) as the backbone of the woodrat classification.

Las ratas cambalacheras del género Neotoma han sido estudiadas en varios tipos de investigaciones incluyendo paleoecología, fisiología, evolución morfológica, sistemática, especiación e hibridación. Recientemente, se han realizado numerosos estudios para elucidar las relaciones evolutivas dentro del género y entre especies cercanamente relacionadas al mismo; en particular la inclusión de nuevos especímenes provenientes de regiones geográficas críticas han brindado nuevas oportunidades para evaluaciones taxonómicas. A partir de estos nuevos datos se realizaron análisis de parsimonia (PARS), Máxima Verosimilitud (MV), e Inferencia Bayesiana (IB) en secuencias de ADN provenientes de nueve genes individuales (cuatro loci mitocondriales: 12S, 16S, CoII, y Cytb; cinco loci nucleares: Adh-I2, Bfib-I7, En2, Mlr, and Myh6) para determinar la relación filogenética de 23 especies de Neotoma. Los resultados de estos análisis presentan una amplia gama de relaciones filogenéticas entre taxa con un soporte nodal importante en los análisis de MV y PARS en algunas posiciones terminales de nivel medio. Varios genes individuales, en particular 12S, Adh-I2, Bfib-I7, CoII, and Cytb, ofrecieron soporte en varias posiciones basales; sin embargo, la resolución filogenética fue reducida en los demás genes. El último análisis de IB, en donde nueve genes se concatenaron en un solo conjunto de datos, produjo soporte en varios clados que correspondieron a especies de grupos previamente reconocidos (floridana, micropus, mexicana, y lepida) y el sub-género Homodontomys. Los niveles de divergencia genética para comparaciones intraespecíficas fluctuaron entre 0.88% (N. magister) y 6.82% (N. fuscipes); para especies hermanas (4.68%­N. devia y N. lepida hasta 12.70%­N. angustapalata y N. nelsoni); y para los miembros de clados cercanos (8.70%­N. bryanti y N. lepida hasta 12.57%­N. goldmani y N. magister). Las evaluaciones de los limites genéricos, subgenéricos y de grupos de especies fueron explorados usando principios filogenéticos en las secuencias de ADN de este trabajo, y también se basaron en las conclusiones morfológicas de estudios previos. Los resultados obtenidos sugieren que la interpretación taxonómica más conservadora incluye el abandono de las delineaciones subgenéricas y se depende en el reconocimiento de ocho grupos de especies (cinerea, floridana, fuscipes, lepida, mexicana, micropus, phenax, y stephensi) como el pilar central de la clasificación de las ratas cambalacheras.

Design and evaluation of neutralizing and fusion inhibitory peptides to Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever (CCHF) is a medically relevant tick-borne viral disease caused by the Bunyavirus, Crimean-Congo hemorrhagic fever virus (CCHFV). CCHFV is endemic to Asia, the Middle East, South-eastern Europe, and Africa and is transmitted in enzootic cycles among ticks, mammals, and birds. Human infections are mostly subclinical or limited to mild febrile illness. Severe disease may develop, resulting in multi-organ failure, hemorrhagic manifestations, and case-fatality rates up to 30%. Despite the widespread distribution and life-threatening potential, no treatments have been approved for CCHF. Antiviral inhibitory peptides, which antagonize viral entry, are licensed for clinical use in certain viral infections and have been experimentally designed against human pathogenic bunyaviruses, with in vitro and in vivo efficacies. We designed inhibitory peptides against CCHFV with and without conjugation to various polyethylene glycol and sterol groups. These additions have been shown to enhance both cellular uptake and antiviral activity. Peptides were evaluated against pseudotyped and wild-type CCHFV via neutralization tests, Nairovirus fusion assays, and cytotoxicity profiling. Four peptides neutralized CCHFV with two of these peptides shown to inhibit viral fusion. This work represents the development of experimental countermeasures for CCHF, describes a nairovirus immunofluorescence fusion assay, and illustrates the utility of pseudotyped CCHFV for the screening of entry antagonists at low containment settings for CCHF.

Transmission of Guanarito and Pirital viruses among wild rodents, Venezuela.

Samples from rodents captured on a farm in Venezuela in February 1997 were tested for arenavirus, antibody against Guanarito virus (GTOV), and antibody against Pirital virus (PIRV). Thirty-one (48.4%) of 64 short-tailed cane mice (Zygodontomys brevicauda) were infected with GTOV, 1 Alston's cotton rat (Sigmodon alstoni) was infected with GTOV, and 36 (64.3%) of 56 other Alston's cotton rats were infected with PIRV. The results of analyses of field and laboratory data suggested that horizontal transmission is the dominant mode of GTOV transmission in Z. brevicauda mice and that vertical transmission is an important mode of PIRV transmission in S. alstoni rats. The results also suggested that bodily secretions and excretions from most GTOV-infected short-tailed cane mice and most PIRV-infected Alston's cotton rats may transmit the viruses to humans.

Genetic evidence for a tacaribe serocomplex virus, Mexico.

We isolated arenavirus RNA from white-toothed woodrats (Neotoma leucodon) captured in a region of Mexico in which woodrats are food for humans. Analyses of nucleotide and amino acid sequence data indicated that the woodrats were infected with a novel Tacaribe serocomplex virus, proposed name Real de Catorce virus.

Crimean-Congo hemorrhagic fever virus in tortoises and Hyalomma aegyptium ticks in East Thrace, Turkey: potential of a cryptic transmission cycle.

BACKGROUND: Recent reports have demonstrated the presence of Crimean-Congo hemorrhagic fever virus (CCHFV) genomic material in Hyalomma aegyptium ticks feeding primarily on tortoises belonging to the genus Testudo. This raises the question if these ticks and their hosts play a role in the natural transmission dynamics of CCHFV. However, the studies are limited, and assessing the relevance of H. aegyptium in perpetuating the virus in nature, and a potential spillover to humans remains unknown. This study aimed to detect CCHFV in H. aegyptium ticks and their tortoise hosts in the East Thrace region of Turkey, where H. aegyptium is the most common human-biting tick and where a high density of tortoises of the genus Testudo can be found. METHODS: During the study period, 21 blood samples from different tortoises (2 T. hermanni and 19 T. graeca), 106 tick pools (containing 448 males, 152 females, 93 nymphs and 60 larvae) collected from 65 tortoises (5 T. hermanni and 60 T. graeca), 38 adult unfed questing ticks (25 males and 13 females, screened individually) and 14 pools (containing 8 nymphs and 266 larvae) of immature unfed questing ticks collected from the ground were screened for CCHFV genome by nested PCR and partial genomes sequenced. RESULTS: As a result of the screening of these 179 samples, 17 (9.5%) were detected as positive as follows: 2 of 21 blood samples (9.52%), 13 (containing 18 nymphs in 3 pools, and 52 males and 8 females in 10 pools) of 106 tick pools from tortoises (12.26%), and 2 of 38 adult questing ticks (5.26%). No positive result was determined in 14 pools of immature questing ticks. CONCLUSIONS: Previous studies have shown that reptiles can participate in the transmission of arthropod-borne viruses, but they may contribute to different aspects of the disease ecology and evolution of tick-borne viral pathogens. Our results indicate the presence of CCHFV in questing and feeding H. aegyptium ticks as well as tortoise hosts. This may indicate that CCHFV circulates in a cryptic transmission cycle in addition to the primary transmission cycle that could play a role in the natural dynamic of the virus and the transmission to humans.

Genetic diversity among Bolivian arenaviruses.

Machupo virus and Chapare virus are members of the Tacaribe serocomplex (virus family Arenaviridae) and etiological agents of hemorrhagic fever in humans in Bolivia. The nucleotide sequences of the complete Z genes, a large fragment of the RNA-dependent RNA polymerase genes, the complete glycoprotein precursor genes, and the complete nucleocapsid protein genes of 8 strains of Machupo virus were determined to increase our knowledge of the genetic diversity among the Bolivian arenaviruses. The results of analyses of the predicted amino acid sequences of the glycoproteins of the Machupo virus strains and Chapare virus strain 200001071 indicated that immune plasma from hemorrhagic fever cases caused by Machupo virus may prove beneficial in the treatment of Bolivian hemorrhagic fever but not hemorrhagic fever caused by Chapare virus.

Diversity among Tacaribe serocomplex viruses (family Arenaviridae) naturally associated with the Mexican woodrat (Neotoma mexicana).

The results of analyses of glycoprotein precursor and nucleocapsid protein gene sequences indicated that an arenavirus isolated from a Mexican woodrat (Neotoma mexicana) captured in Arizona is a strain of a novel species (proposed name Skinner Tank virus) and that arenaviruses isolated from Mexican woodrats captured in Colorado, New Mexico, and Utah are strains of Whitewater Arroyo virus or species phylogenetically closely related to Whitewater Arroyo virus. Pairwise comparisons of glycoprotein precursor sequences and nucleocapsid protein sequences revealed a high level of divergence among the viruses isolated from the Mexican woodrats captured in Colorado, New Mexico, and Utah and the Whitewater Arroyo virus prototype strain AV 9310135, which originally was isolated from a white-throated woodrat (Neotoma albigula) captured in New Mexico. Conceptually, the viruses from Colorado, New Mexico, and Utah and strain AV 9310135 could be grouped together in a species complex in the family Arenaviridae, genus Arenavirus.

Catarina virus, an arenaviral species principally associated with Neotoma micropus (southern plains woodrat) in Texas.

The purpose of this study was to define the taxonomic relationship of an arenavirus principally associated with the southern plains woodrat (Neotoma micropus) in southern Texas to other New World arenaviruses. The results of independent analyses of glycoprotein precursor amino acid sequences and nucleocapsid protein amino acid sequences indicated that the arenavirus in southern Texas is novel (proposed species name Catarina virus) and phylogenetically most closely related to Whitewater Arroyo virus, which is principally associated with the white-throated woodrat (Neotoma albigula) in northwestern New Mexico. Together, the close phylogenetic relationship between Catarina virus and Whitewater Arroyo virus and the association of these viral species with congeneric rodent species support the notion that the principal host relationships of some New World arenaviruses are a product of a long-term shared evolutionary relationship between the virus family Arenaviridae and the rodent family Cricetidae.

Muleshoe Virus and Other Hantaviruses Associated with Neotomine or Sigmodontine Rodents in Texas.

The broad objective of this study was to increase our knowledge of Muleshoe virus and other hantaviruses associated with cricetid rodents in Texas. Anti-hantavirus antibody was found in 38 (3.2%) of 1171 neotomine rodents and 6 (1.8%) of 332 sigmodontine rodents from 10 Texas counties; hantaviral RNA was detected in 23 (71.9%) of 32 antibody-positive rodents. Analyses of nucleocapsid protein gene sequences indicated Muleshoe virus infection in four hispid cotton rats (Sigmodon hispidus) from northern Texas; Bayou virus, three Texas marsh oryzomys (Oryzomys texensis) from the Gulf Coast; Limestone Canyon virus, five brush mice (Peromyscus boylii) from western Texas; and Sin Nombre virus-five Texas mice (P. attwateri), one Lacey's white-ankled deer mouse (P. laceianus), four white-footed mice (P. leucopus), and one fulvous harvest mouse (Reithrodontomys fulvescens) from northern, central, or southern Texas. The results of this study together with the results of a previous study revealed that Muleshoe virus, perhaps in association with S. hispidus, is distributed across northern Texas. Finally, the results of Bayesian analyses of glycoprotein precursor (GPC) gene sequences and pairwise comparisons of complete GPC (amino acid) sequences strengthened support for the notion that Muleshoe virus is distinct from Black Creek Canal virus, Bayou virus, and all other species included in the Bunyaviridae, genus Hantavirus.

Genomic Characterization of Crimean-Congo Hemorrhagic Fever Virus in Hyalomma Tick from Spain, 2014.

Crimean-Congo hemorrhagic fever (CCHF) is a severe tick-borne disease caused by CCHF virus (CCHFV). Ticks in the genus Hyalomma are the main vectors and reservoirs of CCHFV. In Spain, CCHFV was first detected in Hyalomma ticks from Cáceres in 2010. Subsequently, two autochthonous CCHF cases were reported in August 2016. In this study, we describe the characterization of the CCHFV genome directly from Hyalomma lusitanicum collected in Cáceres in 2014. Phylogenetic analyses reveal a close relationship with clade III strains from West Africa, with an estimated divergence time of 50 years. The results of this work suggest that CCHFV has been circulating in Spain for some time, and most likely originated from West Africa.

Catacamas virus, a hantaviral species naturally associated with Oryzomys couesi (Coues' oryzomys) in Honduras.

The primary objective of this study was to extend our knowledge of the geographical distribution, genetic diversity, and natural host associations of the hantaviruses indigenous to North America. Antibody to a hantavirus was found in 5 (20.8%) of 24 Coues' oryzomys (Oryzomys couesi) and none of 41 other rodents captured near the town of Catacamas in eastern Honduras, and a hantavirus was isolated from one of the antibody-positive Coues' oryzomys. Analyses of nucleotide and amino acid sequence data indicated that the viral isolate is a strain of a novel hantaviral species (proposed species name "Catacamas virus") that is phylogenetically most closely related to Bayou virus, a hantaviral species that is principally associated with Oryzomys palustris (marsh oryzomys) in the southeastern United States. Catacamas virus is the first evidence for the occurrence of a hantaviral species in Honduras and the first evidence that a hantaviral species is naturally associated with an Oryzomys species other than O. palustris.

Phylogenetic Relationship of Necoclí Virus to Other South American Hantaviruses (Bunyaviridae: Hantavirus).

The results of a previous study suggested that Cherrie's cane rat (Zygodontomys cherriei) is the principal host of Necoclí virus (family Bunyaviridae, genus Hantavirus) in Colombia. Bayesian analyses of complete nucleocapsid protein gene sequences and complete glycoprotein precursor gene sequences in this study confirmed that Necoclí virus is phylogenetically closely related to Maporal virus, which is principally associated with the delicate pygmy rice rat (Oligoryzomys delicatus) in western Venezuela. In pairwise comparisons, nonidentities between the complete amino acid sequence of the nucleocapsid protein of Necoclí virus and the complete amino acid sequences of the nucleocapsid proteins of other hantaviruses were ≥8.7%. Likewise, nonidentities between the complete amino acid sequence of the glycoprotein precursor of Necoclí virus and the complete amino acid sequences of the glycoprotein precursors of other hantaviruses were ≥11.7%. Collectively, the unique association of Necoclí virus with Z. cherriei in Colombia, results of the Bayesian analyses of complete nucleocapsid protein gene sequences and complete glycoprotein precursor gene sequences, and results of the pairwise comparisons of amino acid sequences strongly support the notion that Necoclí virus represents a novel species in the genus Hantavirus. Further work is needed to determine whether Calabazo virus (a hantavirus associated with Z. brevicauda cherriei in Panama) and Necoclí virus are conspecific.

Epizootiology of Tacaribe serocomplex viruses (Arenaviridae) associated with neotomine rodents (Cricetidae, Neotominae) in southern California.

The objective of this study was to advance our knowledge of the epizootiology of Bear Canyon virus and other Tacaribe serocomplex viruses (Arenaviridae) associated with wild rodents in California. Antibody (immunoglobulin G [IgG]) to a Tacaribe serocomplex virus was found in 145 (3.6%) of 3977 neotomine rodents (Cricetidae: Neotominae) captured in six counties in southern California. The majority (122 or 84.1%) of the 145 antibody-positive rodents were big-eared woodrats (Neotoma macrotis) or California mice (Peromyscus californicus). The 23 other antibody-positive rodents included a white-throated woodrat (N. albigula), desert woodrat (N. lepida), Bryant's woodrats (N. bryanti), brush mice (P. boylii), cactus mice (P. eremicus), and deer mice (P. maniculatus). Analyses of viral nucleocapsid protein gene sequence data indicated that Bear Canyon virus is associated with N. macrotis and/or P. californicus in Santa Barbara County, Los Angeles County, Orange County, and western Riverside County. Together, analyses of field data and antibody prevalence data indicated that N. macrotis is the principal host of Bear Canyon virus. Last, the analyses of viral nucleocapsid protein gene sequence data suggested that the Tacaribe serocomplex virus associated with N. albigula and N. lepida in eastern Riverside County represents a novel species (tentatively named "Palo Verde virus") in the genus Arenavirus.

Phylogeny of the Venezuelan arenaviruses.

Guanarito virus (the etiologic agent of Venezuelan hemorrhagic fever, VHF) and Pirital virus coexist in the region of Venezuela in which Venezuelan hemorrhagic fever is endemic. The purpose of this study was to extend our knowledge of the evolutionary relationship between these two arenaviruses. We determined that the large genomic segments of Guanarito virus and Pirital virus are similar in size and identical in structural organization to the large genomic segments of other South American arenaviruses. For example, the Z proteins and RNA-dependent RNA polymerases of Guanarito virus and Pirital virus are encoded in nonoverlapping open reading frames of opposite polarities. Phylogenetic analyses of Z protein gene nucleotide sequences and RNA-dependent RNA polymerase gene nucleotide sequences grouped Pirital virus with Pichindé virus (a South American arenavirus which, like Pirital virus, does not appear to be pathogenic for humans) and placed the Pirital-Pichindé lineage in a sister relationship to a lineage represented by Guanarito virus and the three other arenaviruses known to cause hemorrhagic fever in South America. These results are concordant with the results of studies on the phylogeny of the arenavirus small genomic segment. Thus, the exchange of genomic elements between Guanarito virus and Pirital virus via recombination or reassortment likely did not contribute to the emergence of Venezuelan hemorrhagic fever.

Maporal virus, a hantavirus associated with the fulvous pygmy rice rat (Oligoryzomys fulvescens) in western Venezuela.

Oryzomine rodents in the southeastern United States, Panama, and southern South America are natural hosts of 6 of the 13 viruses known to cause hantavirus pulmonary syndrome. The purpose of this study was to extend our knowledge of the geographical distribution and genetic diversity of the hantaviruses associated with oryzomine rodents in South America. An infectious hantavirus was isolated from two fulvous pygmy rice rats captured in western Venezuela. Analyses of complete nucleocapsid protein and glycoprotein precursor sequences indicated that the isolates are strains of a novel hantavirus (proposed name "Maporal") which is phylogenetically most closely related to the viruses known to cause hantavirus pulmonary syndrome in southern South America.

Diversity among Tacaribe serocomplex viruses (Family Arenaviridae) associated with the southern plains woodrat (Neotoma micropus).

The southern plains woodrat (Neotoma micropus) is the principal host of Catarina virus in southern Texas and a natural host of other North American Tacaribe serocomplex viruses. The objectives of this study were to increase our knowledge of the genetic diversity among Tacaribe serocomplex viruses associated with N. micropus and to define better the natural host relationships of these viruses. Pairwise comparisons of complete glycoprotein precursor gene sequences and complete nucleocapsid protein gene sequences revealed a high level of genetic diversity among Tacaribe serocomplex viruses associated with N. micropus in western Oklahoma, southern New Mexico, and northern and southern Texas. Collectively, the results of Bayesian analyses of nucleotide sequences and pairwise comparisons of amino acid sequences confirmed that the arenaviruses associated with N. micropus in Oklahoma and New Mexico should be included in the Whitewater Arroyo species complex, and indicated that that the arenaviruses associated with N. micropus in northern Texas are strains of a novel arenaviral species--tentatively named "Middle Pease River virus". Together, the results of assays for arenavirus and assays for anti-arenavirus antibody in 54 southern plains woodrats and 325 other rodents captured at 2 localities suggested that the southern plains woodrat is the principal host of Middle Pease River virus in northern Texas.

Ecology of Catarina virus (family Arenaviridae) in southern Texas, 2001-2004.

A total of 3941 rodents were captured during a 46-month prospective (mark-recapture) study on the ecology of Catarina virus in southern Texas. Antibody reactive against Catarina virus was found in 73 (11.9%) of 611 southern plains woodrats (Neotoma micropus) and none of 3330 other rodents; strains of Catarina virus were isolated from 6 antibody-negative and 9 antibody-positive southern plains woodrats; and the infections in at least 3 southern plains woodrats were chronic. These results affirm the notion that the southern plains woodrat is the principal host of Catarina virus and suggest that Catarina virus infection is highly specific to N. micropus.