M. Kariuki Njenga

Metapneumoviruses in birds and humans

Avian pneumovirus (APV, Turkey rhinotracheitis virus) and Human metapneumovirus (hMPV) are pathogens of birds and humans, respectively, that are associated with upper respiratory tract infections. Based on their different genomic organization and low level of nucleotide (nt) and amino acid (aa) identity with paramyxoviruses in the genus Pneumovirus, APV and hMPV have been classified into a new genus referred to as Metapneumovirus. First isolated in 1970s, APV strains have since been isolated in Europe, Africa, middle east, and United States (US) and classified in four subgroups, APV/A, APV/B, APV/C, and APV/D based on nt and predicted aa sequence identity. Although it was first isolated in 2001, serological evidence indicates that hMPV may have been present in human population from as early as the 1950s. There is only one subgroup of hMPV so far, whose nt and aa sequence identity indicates that it is more closely related to APV/C than to APV/A, APV/B, or APV/D.

Molecular Epidemiology of Subgroup C Avian Pneumoviruses Isolated in the United States and Comparison with Subgroup A and B Viruses

ABSTRACT The avian pneumovirus (APV) outbreak in the United States is concentrated in the north-central region, particularly in Minnesota, where more outbreaks in commercial turkeys occur in the spring (April to May) and autumn (October to December). Comparison of the nucleotide and amino acid sequences of nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), and second matrix (M2) genes of 15 U.S. APV strains isolated between 1996 and 1999 revealed between 89 and 94% nucleotide sequence identity and 81 to 95% amino acid sequence identity. In contrast, genes from U.S. viruses had 41 to 77% nucleotide sequence identity and 52 to 78% predicted amino acid sequence identity with European subgroup A or B viruses, confirming that U.S. viruses belonged to a separate subgroup. Of the five proteins analyzed in U.S. viruses, P was the most variable (81% amino acid sequence identity) and N was the most conserved (95% amino acid sequence identity). Phylogenetic comparison of subgroups A, B, and C viruses indicated that A and B viruses were more closely related to each other than either A or B viruses were to C viruses.

A Wild-Type Porcine Encephalomyocarditis Virus Containing a Short Poly(C) Tract Is Pathogenic to Mice, Pigs, and Cynomolgus Macaques

ABSTRACT Previous studies using wild-type Encephalomyocarditis virus (EMCV) and Mengo virus, which have long poly(C) tracts (61 to 146 Cs) at the 5′ nontranslated region of the genome, and variants of these viruses genetically engineered to truncate or substitute the poly(C) tracts have produced conflicting data on the role of the poly(C) tract in the virulence of these viruses. Analysis of the nucleotide sequence of an EMCV strain isolated from an aborted swine fetus (EMCV 30/87) revealed that the virus had a poly(C) tract that was 7- to 10-fold shorter than the poly(C) tracts of other EMCV strains and 4-fold shorter than that of Mengo virus. Subsequently, we investigated the virulence and pathogenesis of this naturally occurring short-poly(C)-tract-containing virus in rodents, pigs, and nonhuman primates. Infection of C57BL/6 mice, pigs, and cynomolgus macaques resulted in similar EMCV 30/87 pathogenesis, with the heart and brain as the primary sites of infections in all three animals, but with different disease phenotypes. Sixteen percent of EMCV 30/87-infected pigs developed acute fatal cardiac failure, whereas the rest of the pigs were overtly asymptomatic for as long as 90 days postinfection (p.i.), despite extensive myocardial and central nervous system (CNS) pathological changes. In contrast, mice infected with ≥4 PFU of EMCV 30/87 developed acute encephalitis that resulted in the death of all animals (n = 25) between days 2 and 7 p.i. EMCV 30/87-infected macaques remained overtly asymptomatic for 45 days, despite extensive myocardial and CNS pathological changes and viral persistence in more than 50% of the animals. The short poly(C) tract in EMCV 30/87 (CUC5UC8) was comparable to that of strain 2887A/91 (C10UCUC3UC10), another recent porcine isolate.

Nucleotide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glycoprotein gene: phylogenetic analysis and molecular epidemiology of U.S. pneumoviruses.

ABSTRACT A serologically distinct avian metapneumovirus (aMPV) was isolated in the United States after an outbreak of turkey rhinotracheitis (TRT) in February 1997. The newly recognized U.S. virus was subsequently demonstrated to be genetically distinct from European subtypes and was designated aMPV serotype C (aMPV/C). We have determined the nucleotide sequence of the gene encoding the cell attachment glycoprotein (G) of aMPV/C (Colorado strain and three Minnesota isolates) and predicted amino acid sequence by sequencing cloned cDNAs synthesized from intracellular RNA of aMPV/C-infected cells. The nucleotide sequence comprised 1,321 nucleotides with only one predicted open reading frame encoding a protein of 435 amino acids, with a predicted Mr of 48,840. The structural characteristics of the predicted G protein of aMPV/C were similar to those of the human respiratory syncytial virus (hRSV) attachment G protein, including two mucin-like regions (heparin-binding domains) flanking both sides of a CX3C chemokine motif present in a conserved hydrophobic pocket. Comparison of the deduced G-protein amino acid sequence of aMPV/C with those of aMPV serotypes A, B, and D, as well as hRSV revealed overall predicted amino acid sequence identities ranging from 4 to 16.5%, suggesting a distant relationship. However, G-protein sequence identities ranged from 72 to 97% when aMPV/C was compared to other members within the aMPV/C subtype or 21% for the recently identified human MPV (hMPV) G protein. Ratios of nonsynonymous to synonymous nucleotide changes were greater than one in the G gene when comparing the more recent Minnesota isolates to the original Colorado isolate. Epidemiologically, this indicates positive selection among U.S. isolates since the first outbreak of TRT in the United States.

More severe neurologic deficits in SJL/J male than female mice following Theiler's virus-induced CNS demyelination.

Although multiple sclerosis (MS) is more prevalent in women than men, male MS patients develop more severe clinical symptoms and deteriorate faster than female patients. We investigated the differences in CNS demyelinating disease between SJL/J male and female mice following Theilers murine encephalomyelitis virus (TMEV) infection. Infected female mice had consistently higher serum levels of virus-specific IgG at 14 and 21 days and and 7 months postinfection, which resulted in less infectious virus in CNS. All male mice infected for 6 to 7 months developed paralysis, with 50% displaying bilateral posterior limb paralysis, whereas 77% of age-matched female mice were paralyzed, all displaying unilateral posterior limb paralysis. Male mice infected for 6 to 7 months performed up to threefold fewer spontaneous horizontal and vertical movements (activity box test) compared to infected age-matched females. In addition, infected male mice performed the coordination and balance (Rotarod) test at 27 +/- 4% of the expected level (expressed as a percentage of that of uninfected age-matched mice), whereas infected female mice performed at 41 +/- 5% of the expected level. Male mice had a small increase in the extent of spinal cord white matter demyelination analyzed at both 45 days and between 6 and 7 months postinfection. For individual male and female mice, the extent of demyelination had a negative linear relationship with the neurologic performances. The emergence of a disease paradigm similar to MS supports using the TMEV model to investigate molecular and genetic factors responsible for the gender dimorphism in MS and other autoimmune diseases.

Absence of Spontaneous Central Nervous System Remyelination in Class II-deficient Mice Infected with Theiler's Virus

We previously showed that Theilers murine encephalomyelitis virus (TMEV)-infected major histocompatibility complex (MHC) class II-deficient mice develop both demyelination and neurologic deficits, whereas MHC class I-deficient mice develop demyelination but no neurologic deficits. The absence of neurologic deficits in the class I-deficient mice was associated with preserved sodium channel densities in demyelinated lesions, a relative preservation of axons, and extensive spontaneous remyelination. In this study, we investigated whether TMEV-infected class II-deficient mice, which have an identical genetic background (C57BL/6 x 129) as the class I-deficient mice, have preserved axons and spontaneous myelin repair following chronic TMEV-infection. Both class I- and class II-deficient mice showed similar extents of demyelination of the spinal cord white matter 4 months after TMEV infection. However, the class I-deficient mice demonstrated remyelination by oligodendrocytes, whereas class II-deficient mice showed minimal if any myelin repair. Demyelinated lesions, characterized by inflammatory infiltrates in both mutants, revealed disruption of axons in class II- but not class I-deficient mice. Further characterization revealed that even though class II-deficient mice lacked TMEV-specific IgG, they had virus-specific IgM, which, however, did not neutralize TMEV in vitro. In addition, class II-deficient mice developed TMEV-specific cytotoxic T-lymphocytes in the CNS during the acute (7 days) disease, but these cytotoxic lymphocytes were not present in the chronic stage of disease, despite a high titer of infectious virus throughout the disease. We envision that the presence of demyelination, high virus titer, absence of remyelination, and axonal disruption in chronically infected class II-deficient mice contributes to the development of paralytic disease.

Short‐term treatment with interferon‐α/β promotes remyelination, whereas long‐term treatment aggravates demyelination in a murine model of multiple sclerosis

The mechanisms by which type I interferons (IFN) reduce the rate and severity of exacerbations in multiple sclerosis are unknown. We utilized a model of multiple sclerosis to determine the extent of demyelination and remyelination in Theilers murine encephalomyelitis virus (TMEV)‐infected SJL/J mice treated with mouse IFN‐α/β for a short (5 weeks) or a long (16 weeks) period. All mice were chronically infected with TMEV to simulate the clinical situation in multiple sclerosis. Short‐term IFN‐α/β treatment increased the percent of remyelinated spinal cord white matter by threefold when compared with phosphate‐buffered saline (PBS) treatment (P < 0.02), but it did not affect the extent of demyelination. In contrast, long‐term IFN‐α/β treatment increased the extent of demyelination by twofold (P < 0.03). Long‐term treatment increased the absolute area of remyelination, but the percent remyelination as a function of area of demyelination was not changed because of increased demyelination. An immunomodulatory mechanism may have contributed to the effect of IFN‐α/β on white matter pathology because treated mice had higher anti‐TMEV IgGs in serum and demonstrated decreased numbers of B and T lymphocytes infiltrating the central nervous system (CNS). There was no correlation between the level of anti‐ IFN‐α/β antibodies and the extent of demyelination or remyelination. These results indicate that the length of type I IFN treatment may have paradoxical effects on demyelination and remyelination. J. Neurosci. Res. 59:661–670, 2000

Direct Comparison of Demyelinating Disease Induced by the Daniel's Strain and BeAn Strain of Theiler's Murine Encephalomyelitis Virus

We compared CNS disease following intracere‐bral injection of SJL mice with Daniels (DA) and BeAn 8386 (BeAn) strains of Theilers murine encephalomyelitis virus (TMEV). In tissue culture, DA was more virulent then BeAn. There was a higher incidence of demyelination in the spinal cords of SJL/J mice infected with DA as compared to BeAn. However, the extent of demyelination was similar between virus strains when comparing those mice that developed demyelination. Even though BeAn infection resulted in lower incidence of demyelination in the spinal cord, these mice showed significant brain disease similar to that observed with DA. There was approximately 100 times more virus specific RNA in the CNS of DA infected mice as compared to BeAn infected mice. This was reflected by more virus antigen positive cells (macrophages/microglia and oligodendrocytes) in the spinal cord white matter of DA infected mice as compared to BeAn. There was no difference in the brain infiltrating immune cells of DA or BeAn infected mice. However, BeAn infected mice showed higher titers of TMEV specific antibody. Functional deficits as measured by Rotarod were more severe in DA infected versus BeAn infected mice. These findings indicate that the diseases induced by DA or BeAn are distinct.

Isolation of avian pneumovirus from mallard ducks that is genetically similar to viruses isolated from neighboring commercial turkeys.

Our earlier studies demonstrating avian pneumovirus (APV) RNA in wild geese, sparrows, swallows, starlings and mallard ducks suggested that wild birds might be involved in the circulation of APV in the United States. To determine whether turkey virus can be transmitted to the free flying birds, we placed APV-negative mallard ducks next to a turkey farm experiencing a severe APV outbreak and in an area with a large population of waterfowls. The sentinel ducks did not develop clinical APV disease but infectious APV (APV/MN-12) was recovered from choanal swabs after 2 weeks, and anti-APV antibodies detected after 4 weeks. Four APV isolates recovered from the neighboring turkeys that were experiencing an APV outbreak at the same time shared 95-99% nucleotide identity and 97-99% predicted amino acid identity with the duck isolate. In addition experimental infection of turkey poults with APV/MN-12 resulted in detection of viral RNA in nasal turbinates and APV-specific IgG in serum. These results indicate that the APV isolates from turkeys and ducks shared a common source, and the viruses from different avian species can cross-infect.

A Wild Goose Metapneumovirus Containing a Large Attachment Glycoprotein Is Avirulent but Immunoprotective in Domestic Turkeys

ABSTRACT The genomic structure and composition of an avian metapneumovirus (aMPV) recently isolated from wild Canada geese (goose 15a/01) in the United States, together with its replication, virulence, and immunogenicity in domestic turkeys, were investigated. The sizes of seven of the eight genes, sequence identity, and genome organization of goose aMPV were similar to those of turkey aMPV subtype C (aMPV/C) strains, indicating that it belonged to the subtype. However, the goose virus contained the largest attachment (G) gene of any pneumovirus or metapneumovirus, with the predicted G protein of 585 amino acids (aa) more than twice the sizes of G proteins from other subtype C viruses and human metapneumovirus and more than 170 aa larger than the G proteins from the other aMPV subtypes (subtypes A, B, and D). The large G gene resulted from a 1,015-nucleotide insertion at 18 nucleotides upstream of the termination signal of the turkey aMPV/C G gene. Three other aMPV isolates from Canada geese had similarly large G genes, whereas analysis of recent aMPV strains circulating in U.S. turkeys did not indicate the presence of the goose virus-like strain. In vitro, the goose virus replicated to levels (2 × 105 to 5 × 105 50% tissue culture infective dose) comparable to those produced by turkey aMPV/C strains. More importantly, the virus replicated efficiently in the upper respiratory tract of domestic turkeys but with no clinical signs in either day-old or 2-week-old turkeys. The virus was also horizontally transmitted to naïve birds, and turkey infections with goose 15a/01 induced production of aMPV-specific antibodies. Challenging day-old or 2-week-old turkeys vaccinated with live goose aMPV resulted in lower clinical scores in 33% of the birds, whereas the rest of the birds had no detectable clinical signs of the upper respiratory disease, suggesting that the mutant virus may be a safe and effective vaccine against aMPV infection outbreaks in commercial turkeys.