The PIVs are enveloped, cytoplasmic viruses (Fig. 34.1) with single-stranded, nonsegmented, negative-sense RNA genomes of 14.9 to 17.3 kb (Fig. 34.2). They are distributed among three genera (namely Respirovirus, Rubulavirus, and Avulavirus)
of subfamily Paramyxovirinae, family Paramyxoviridae, order Mononegavirales.²¹⁸ (The other subfamily is Pneumovirinae, which contains HRSV, human metapneumovirus [HMPV], and their relatives). HPIV1 and HPIV3, and their respective murine and bovine relatives SeV and BPIV3, constitute the genus Respirovirus (Fig. 34.3). HPIV2, its relatives PIV5 and SV41, and HPIV4 are part of the genus Rubulavirus. Rubulavirus is a diverse genus that also contains related viruses that are not considered PIVs, such as MuV, Mapuera virus, and porcine rubulavirus. The various APMV serotypes constitute the genus Avulavirus.

The relationships between the PIVs are illustrated in Figure 34.3 by alignment of the amino acid sequences of the L proteins. Representative viruses from Morbillivirus and Henipavirus, two other genera of the subfamily Paramyxovirinae, are included for comparison. This shows that the PIVs as a whole are broadly divergent and are not clearly demarcated by sequence relatedness as a group distinct from the non-PIV members of Paramyxovirinae. This comparison also shows the close relatedness between some of the human and animal PIVs, such as between HPIV1 and SeV and between HPIV3 and BPIV3. It is likely that these closely related viruses arose from transmission across host species.

The relationships between the PIVs also are illustrated in Tables 34.1 to 34.3 by the percent amino acid sequence identity for the two major surface antigens, namely the fusion F glycoprotein (Table 34.1) and the hemagglutinin-neuraminidase HN glycoprotein (Table 34.2), as well as for the large polymerase L protein (Table 34.3). This illustrates, for example, that the HPIV serotype distinctions are associated with
amino acid sequence identities of less than 50% for F and HN. The APMV serotypes are not shown in these tables, but they also usually (but not always) have less than 50% amino acid sequence identity between F and HN of the different serotypes.⁴³,¹⁷²,²¹³,²¹⁴,²⁶⁵,³¹⁸,³⁵³,⁴⁰¹,⁴⁰² For example, the percent amino acid sequence identity between APMV5 versus serotypes 1, 2, 3, 4, 6, 7, 8, and 9 is, respectively: 41, 47, 31, 33, 55, 37, 46, and 37 for the F protein; and 35, 42, 33, 30, 56, 43, 41, and 31 for the HN protein.³¹⁹

Antigenic reactivity based on binding assays can be detected between PIVs within a genus with polyclonal sera and, less frequently, with monoclonal antibodies (MAbs).¹⁶⁷,²⁰³,²⁷¹ A lower level of reactivity between genera sometimes is detected with polyclonal sera, although there is no group antigen encompassing the three PIV genera.

HPIV4 has been segregated into two variants, A and B, based on antigenic differences detected by hemadsorption-inhibition (HI) and MAb reactivity.²⁰⁴ Sequence analysis shows that these two subgroups are very closely related: the percent identity between the F, HN, and L proteins is 95, 87, and 97, respectively (Tables 34.1 to 34.3), and they likely would not be distinguishable in neutralization assays with postinfection sera. Variation within the other HPIV serotypes appears to be somewhat less. For HPIV2, the percent amino acid sequence

identity between the V94 strain versus the V98 and Greer strains was, respectively, 98 and 99 for F, 95 and 96 for HN, and 99 and nearly 100 for L.³³⁷ For HPIV3, comparison of the F protein sequence of prototype strain Washington/47885/57 with seven clinical strains revealed 98% or more identity,⁶² and comparison of HN with six clinical strains revealed 97% or more identity.³⁸⁵ For HPIV1, comparison of 40 strains showed that the percent amino acid sequence identity for the HN protein was 95% or greater.¹⁴⁶ For NDV, comparison of 50 strains from various times and places of isolation showed that the percent identity between F and HN was 91% and 90% or greater, respectively.³¹⁷ Some of the other animal PIVs, such as APMV2,³⁵² APMV3,²¹⁴ APMV6,⁴⁰² and BPIV3,¹⁵² have been found to have somewhat greater diversity (i.e., intraserotype amino acid sequence identity for F and HN of 75% to 79% for APMV2, 70% to 73% for APMV3, 81% to 86% for APMV6, and 86% to 89% for BPIV3), resulting in distinct genotypes or subgroups within a serotype. In the case of APMV2, APMV3, and APMV6, this has been shown to be associated with a modest degree of antigenic difference detectable with postinfection sera.

NDV is notable because the many highly related naturally occurring isolates and strains that have been recovered for this single serotype exhibit a broad spectrum of virulence, ranging from nonvirulent or mildly virulent (lentogenic), to moderately virulent (mesogenic), to highly virulent (velogenic). Lentogenic strains are associated with subclinical infection or can cause mild respiratory tract disease, and the more attenuated natural isolates are used as live vaccines. At the other extreme, velogenic strains can be highly virulent and, depending on the strain, can cause hemorrhagic lesions in the intestines (viscerotropic) or neurologic disease (neurotropic).³¹⁷ In contrast, there is no evidence of differences in virulence among the various isolates of each of the four serotypes of HPIV, although this has not been studied extensively. Little is known about the possible diversity of disease within the other animal PIVs.

The virions of PIVs are medium-sized particles of 150 to 200 nm. Fixed, negatively stained virions typically appear in electron micrographs as pleomorphic (irregularly shaped) round particles (Fig. 34.1).⁵⁸,¹⁵⁶,²⁵³,³²⁴,³⁶⁹ Filamentous virions have been described in some cases, such as for HPIV2.⁴⁰⁵ Cryoelectron microscopy of ice-embedded SeV and PIV5 provided images of virions as predominantly perfect spheres of varied diameters.¹⁵³,²³³,³⁶⁹ This suggests that the irregular shapes of particles observed with conventional electron microscopy are artifacts of sample fixation and dehydration, whereas the variations in size were observed using both methods and thus may be authentic. Seventy-one percent of the ice-embedded PIV5 virions consisted of spheres of 129 to 360 nm (average 217 nm), and the remainder were elongated particles of up to 445 nm.³⁶⁹

PIVs replicate in the cytoplasm and bud through the plasma membrane (Fig. 34.1B). The virion consists of a nucleocapsid that is packaged in a lipid envelope derived from the host cell plasma membrane during budding (Fig. 34.1). In the nucleocapsid, the viral genome is tightly bound along its entire length with the nucleoprotein N at a ratio of one protein molecule per six nucleotides (2,484 to 2,877 protein molecules, depending on the length of the viral genome). Associated with the nucleocapsid in the virus particle are approximately 300 copies of the phosphoprotein P and approximately 40 copies of the major polymerase protein L, based on studies with SeV.²⁰⁰,²¹⁹ Rubulaviruse virions contain an additional nucleocapsid-associated protein called V,²⁸⁴ and the virion of the Respirovirus SeV has been reported to contain 40 copies of a small protein called C, also associated with the nucleocapsid.⁴⁰³ Electron micrographs of nucleocapsids released from PIV virions indicate a length of approximately 1.0 to 1.1 μm.⁶⁵,²³³ The nucleocapsid with its associated proteins has RNA-dependent RNA polymerase activity.¹³⁴ Purified virions can be activated for cell-free transcription by disruption of the envelope with detergent and can transcribe the viral genome in its entirety into messenger RNAs (mRNAs).⁶⁴

The envelope bears spike-like surface projections composed of homotrimers and tetramers of the F and HN glycoproteins, respectively. Based on cryoelectron microscopy, PIV5 virions were estimated to contain approximately 2,000 glycoprotein spikes per 200 nm particle, with an average spike length of 14.2 nm.³⁶⁹ PIV5 and AMPV6 have a third, small transmembrane protein, SH (“small hydrophobic”). The nonglycosylated matrix M protein is associated with the inner surface of the envelope. The hemagglutination activity of the HN protein mediates adsorption of virus to the host cell to initiate infection. The cellular receptor for the PIVs is N-acetylneuraminic
acid (sialic acid) in a terminal linkage to cellular glycoproteins and glycolipids.³⁵⁶,⁴¹¹ In the case of HPIV3, cell surface nucleolin also has been reported to serve as a receptor co-factor.²⁹ Viral attachment can be measured experimentally by the agglutination of erythrocytes by virus in suspension (hemagglutination) or by the adsorption of erythrocytes to infected cell monolayers expressing HN (hemadsorption) as was used in the original detection of the HPIVs. Late in infection, the neuraminidase activity of HN cleaves sialic acid to facilitate release of progeny virions. Neuraminidase activity can be quantified using sialic acid derivatives as substrates in a colorimetric or fluorometric assay. The F protein mediates fusion between the viral envelope and the host cell plasma membrane, an activity that can be measured in vitro by lysis of erythrocytes (hemolysis).

The PIV genome is a single strand of negative-sense RNA that ranges in length from 14,904 (APMV2) to 17,262 (APMV5) nucleotides (nt). The differences in genome length between the different PIVs are mostly due to differences in the lengths of noncoding sequences rather than substantial differences in the lengths of open reading frames (ORFs). The PIV genome is not capped or polyadenylated. It contains, in 3′ to 5′ order: a short 3′ extragenic leader region of 55 nt (except in the case of HPIV2, for which the leader region is 70 nt), followed by six genes encoding the N, P, M, F, HN, and L proteins, followed by an extragenic trailer region of 21 to 291 nt (Fig. 34.2; note that the longest, 291-nt trailer region is that of APMV-3 and is not shown in this figure). Sequences of the leader regions of selected PIVs are shown in e-Fig. 34.1A. As noted below, the P gene also encodes one or more accessory proteins—namely C, V, W, I, and D—depending on the virus (Fig. 34.2). PIV5 and APMV-6 each contain a seventh small gene that is located between F and HN and encodes the SH protein. In the case of Respirovirus, the PIV genes are separated by intergenic (IG) regions that are conserved trinucleotides (usually 3′-GAA in genome-sense); in the case of Rubulavirus and Avulavirus the IG regions have nonconserved sequences of variable length (0 to 183 nt) (Fig. 34.2; also, see e-Fig. 34.1B for gene junction sequences of selected PIVs).

Transcription and RNA replication occur in the cytoplasm and follow the Mononegavirales model. Briefly, the genes are transcribed sequentially in their 3′ to 5′ order to yield separate nonoverlapping mRNAs that are polyadenylated, capped, and methylated. RNA synthesis also yields short nonpolyadenylated and noncapped transcripts of the leader and trailer regions. Transcription is guided by short conserved gene-start (GS) and gene-end (GE) transcription signals that flank each gene (see e-Fig. 34.1B for gene junction sequences of selected PIVs). For RNA replication, the polymerase ignores the GS and GE signals and produces a complete positive-sense copy of the genome that is called the antigenome. Like the genome, the antigenome is not capped or polyadenylated. Both the genome and antigenome are completely bound with N protein.¹⁹⁹ Encapsidation of nascent genomes and antigenomes is thought to drive chain elongation during RNA replication. The tightly encapsidated nature of the nucleocapsid likely shields the uncapped and nonpolyadenylated genome/antigenome from degradation. It also likely shields the genome/antigenome from recognition by the cytoplasmic helicases retinoic acid-inducible gene 1 (RIG-I) and Melanoma Differentiation-Associated protein 5 (MDA5), which detect triphosphorylated RNA and double-stranded RNA (dsRNA) and initiate signaling to activate the cellular transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB) to induce type I interferon (IFN) and proinflammatory cytokines. This also reduces activation of protein kinase R (PKR), which is triggered through dsRNA to activate NF-κB as well as to phosphorylate eukaryotic translation initiation factor eIF-2a and thereby inhibit translational initiation as part of host defense. As another example of how viral RNA can affect host cell responses, one of the products of SeV RNA replication is a 55-nt aborted RNA representing the 5′ trailer region that contains a U-rich sequence that inhibits apoptosis by binding to the proapoptotic factor T-cell intracellular antigen 1 related (TIAR).¹⁶⁶

The nucleotide lengths of the genomes and antigenomes of the PIVs (and of all of subfamily Paramyxovirinae) are even multiples of six. This property is essential for efficient RNA replication and is called the “rule of six”.¹⁹⁹–²⁰⁰,²⁰¹,³³⁷ This is thought to reflect an obligatory nucleocapsid organization in which each N protein monomer associates with exactly six nucleotides. In experiments to recover recombinant HPIV2 and HPIV3 viruses whose nucleotide lengths were designed to not be even multiples of six, the recovered viruses contained genomes that had mutated to conform to the rule.³³⁶,³³⁷

Each PIV gene encodes—via transcribed mRNA—a single major protein, with the exception of the P gene that can encode additional proteins in two ways that are described briefly here and in greater detail for the HPIVs in e-Fig. 34.2. First, all of the PIVs in the genus Respirovirus contain a C ORF that initiates near the 5′ end of the P mRNA, closely overlapping the start of the P ORF. Depending on the virus, the C ORF has from one to four different translational start sites that are utilized to give rise to up to four carboxy–co-terminal C proteins. Rubulavirus and Avulavirus do not have a C ORF. Second, the P genes of most PIVs encode additional proteins by “RNA editing”.¹⁹⁹,²⁰¹,³⁸⁸ This involves the co-transcriptional insertion of 1 or more G residues into the nascent mRNA by polymerase stuttering at an editing motif midway along the P gene. An array of mRNAs is produced: they include the unedited form as well as subpopulations that contain 1, 2, or more G residues inserted at the editing site. The insertion of 1 G residue (or 3+1, and so on) or 2 residues (or 3+2, and so on) creates frameshifts that access ORFs in the two other reading frames. For the PIVs of Respirovirus and Avulavirus, the unedited mRNA encodes the P protein,²⁸⁶,³⁴⁸,³⁸⁸ and the addition of a single G by RNA editing fuses the upstream half of the P ORF to an internal ORF encoding a domain with a conserved cysteine-rich domain: the resulting protein is called V. The addition of 2 G residues fuses the upstream half of the P ORF to an ORF in the third reading frame: this downstream ORF encodes only a few added amino acids and results in a protein called W, except in the case of HPIV3 and BPIV3 in which the number of added amino acids is substantially more and the resulting protein is called D (e-Fig. 34.2). HPIV1 is an exception because it does not appear to engage in RNA editing.²⁴⁵,³⁰⁷ In addition, although HPIV3 does engage in RNA editing, the V ORF is separated from the editing site by two or more (depending on the strain) stop codons in the same reading frame that may preclude expression of V (see e-Fig. 34.2).¹⁰⁸ For the PIVs of Rubulavirus, the exact-copy mRNA encodes the V protein, whereas an edited version containing
two inserted G residues encodes P.¹⁹²,²⁰⁸,²⁷⁶,³⁴²,³⁷⁰ An edited version containing one inserted residue encodes the I protein, which is the Rubulavirus equivalent of W. [See the ebook for more information on coding assignments and RNA editing.)

Several factors control the relative efficiency of transcription of the various PIV genes. As is typical for Mononegavirales, there is a gradient of transcription in which promoter-proximal genes are expressed somewhat more efficiently than promoter-distal genes.⁶⁴,¹³⁸ This is thought to be due to polymerase fall-off at the gene junctions.¹⁶⁸ However, with the exception of L, the gradient of expression is not continuous or steep; in the case of SeV, for example, the P, M, F and HN mRNAs accumulate at 0.30, 1.15, 0.61, and 0.38 times the level of N.¹⁴⁸ Accumulation of the L mRNA is much lower (0.02 that of N). Differences in transcription signals also influence transcription. In PIV5, the efficiency of transcription across the different gene junctions, measured by the relative level of expression of the downstream versus upstream gene, was found to vary over a fourfold range, indicative of regulation at the level of the termination/re-initiation at the gene junctions.¹³⁸ However, the HN-L junction was not associated with a particularly high level of fall-off.¹³⁸ This suggests that the low level of expression of L relative to the other genes is due to some other factor such as polymerase fall-off during L gene transcription or instability of the L mRNA.

A number of PIVs have evolved mechanisms for downregulating expression of the F gene. The M GE signal of HPIV3 contains an apparent eight-nucleotide insertion that causes increased M-F readthrough (see e-Fig. 34.1B).³⁴⁴ The M GE signal of PIV5 contains a single nucleotide substitution that has the same effect.³⁰⁰ In SV41, the M GE signal is lacking altogether and M is expressed solely as an M-F readthrough mRNA.³⁷⁶ Interestingly, the F gene of SV41 also is expressed as a monocistronic mRNA by initiation at its GS signal, but this occurs at a reduced level because the majority of the polymerase molecules are already engaged in reading across the M-F junction. In HPIV1, the same effect of increased production of M-F mRNA at the expense of monocistronic F mRNA was observed, and studies with recombinant viruses mapped the effect to a combination of features, namely the intergenic sequence, the F GS signal, and the long upstream nontranslated region of the F gene.³¹ Therefore, various features in these different viruses result in the synthesis of an M-F readthrough mRNA at the expense of a monocistronic F mRNA. In these M-F readthrough mRNAs, the F ORF would not be efficiently accessed by ribosomes due to its internal position.²¹¹ Finally, SeV downregulates expression of its F gene by yet another mechanism, namely through a suboptimal GS signal.¹⁸⁸ Therefore, each of these strategies results in reduced expression of this fusogenic factor. In the case of SeV, this was shown to reduce the virulence of the virus.¹⁸⁸ It might be that, by reducing morbidity and mortality in the host, the virus increases its opportunities for shedding and spread.

All PIVs encode six common proteins: N, P, M, F, HN, and L, all of which are essential for virus replication. All members encode at least one additional protein from the P gene (C, V, D, W, and I, depending on the virus). PIV5 and APMV-6 also encode a small hydrophobic transmembrane SH protein.

Postinfection sera from animals and humans contain antibodies against most or all of the major PIV proteins. However, the HN and F proteins are the only antigens that have been shown to induce antibodies that neutralize infectivity, and they have been shown to be major independent protective antigens. In vivo, the parenteral administration of polyclonal or monoclonal antibodies specific to SeV HN or F mediated resistance to challenge with SeV.³⁰² Sera obtained from children following HPIV3 infection that contain antibodies specific to the HN and F proteins have been shown to have virus-neutralizing activity.¹⁸⁵ Infection of rodents with vaccinia virus recombinants expressing the HPIV3 HN or F glycoprotein, or immunization with purified HN and F glycoprotein, showed that either protein induced a high level of resistance to HPIV3 challenge, with HN being more protective than F.⁷,³³,³⁰³,³⁴⁵

The “internal” PIV proteins also induce a protective response. This was demonstrated in experiments in hamsters using a recombinant version of HPIV3 in which the HN and F surface antigen genes were replaced by those of HPIV1. This made it possible to compare the relative contributions of the “internal” proteins and the surface glycoproteins to protection.³⁶³ The HN and F proteins induced a high level of protection (in this case specific to HPIV1) that was long-lived. In contrast, the HPIV3-specific protection attributed to the internal proteins—which presumably was mediated by major histocompatibility class I-restricted, CD8+ cytotoxic T lymphocytes (CTLs)—was weaker and waned over a period of several months.³⁶³ This suggests that cellular immunity can contribute significantly to protection for a short period following infection, but is not effective in providing long-term protection.